Benzamide or benzamine compounds useful as anticancer agents for the treatment of human cancers

ABSTRACT

The described invention provides small molecule anti-cancer compounds for treating tumors that respond to cholesterol biosynthesis inhibition. The compounds selectively inhibit the cholesterol biosynthetic pathway in tumor-derived cancer cells, but do not affect normally dividing cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication No. 62/189,069 filed on Jul. 6, 2015, the entire contents ofwhich are incorporated by reference herein.

FIELD OF THE INVENTION

The described invention relates to small molecule anti-cancertherapeutics.

BACKGROUND OF THE INVENTION

Gliomas

Glial cells, the most abundant cell type in the central nervous system,are cells that surround neurons and provide support for and insulationbetween them. Unlike neurons, glial cells do not conduct electricalimpulses. There are two major classes of glial cells in the centralnervous system: astrocytes and oligodendrocytes (Kandel E R, et al.,Principles of Neural Science, 4th Ed. McGraw-Hill New York (2000), Ch.2, pp. 20-21).

Glial cells in the vertebrate nervous system are divided into two majorclasses: microglia and macroglia. Microglia are phagocytes that aremobilized after infection, injury or disease, which arise frommacrophages outside the nervous system. Three types of macroglial cellspredominate in the vertebrate nervous system: oligodendrocytes, Schwanncells, and astrocytes. Astrocytes, the most numerous of glial cells inthe central nervous system characterized by their star-like shape andthe broad end-feet on their processes, are thought to play a nutritiverole, and help form an impermeable lining in the brains capillaries andvenules—the blood brain barrier—that prevents toxic substances in theblood from entering the brain. Oligodendrocytes, small cells withrelatively few processes, and Schwann cells produce the myelin used toinsulate nerve cell axons.

The term “glioma” encompasses all tumors thought to originate in theglial cell linage. (Veliz, I. et al., “Advances and challenges in themolecular biology and treatment of glioblastoma—is there any hope forthe future?” Ann. Trans. Med. 3(1): 7. Doi:10.3978/j.issn.2305-5939.2014.10.06. The location of the tumor dependson the type of cells from which it originates.

Malignant gliomas exhibit properties that resemble astrocytes oroligodendrocytes, hence the designation as astrocytomas andoligodendrogliomas. These tumors are graded on a scale from I to IV,based on how normal or abnormal the cells look. Of numerous gradingsystems in use, the most common is the World Health Organization (WHO)grading system for glioma (Louis D N, et al., Acta Neuropathol, 2007,114(2):97-109). Grade I tumors are slow-growing, nonmalignant, andassociated with long-term survival. Grade II tumors are relativelyslow-growing but sometimes recur as higher grade tumors. They can benonmalignant or malignant. Grade III tumors are malignant and oftenrecur as higher grade tumors. Grade IV tumors reproduce rapidly and arevery aggressive malignant tumors.

Low grade astrocytomas usually are localized and grow slowly. High gradeastrocytomas grow at a rapid pace and are infiltrative. Astrocytomas canappear in various parts of the brain and nervous system, including thecerebellum, the cerebrum, the central areas of the brain, the brainstem,and the spinal cord.

Pilocytic Astrocytoma (also called Juvenile Pilocytic Astrocytoma), aregrade I astrocytomas, which typically stay in the area where theystarted and do not spread. They are considered the “most benign”(noncancerous) of all the astrocytomas. Two other, less well known gradeI astrocytomas are cerebellar astrocytoma and desmoplastic infantileastrocytoma.

Diffuse Astrocytoma (also called Low-Grade or Astrocytoma Grade II)(e.g., Fibrillary, Gemistocytic, Protoplasmic Astrocytoma) tend toinvade surrounding tissue and grow at a relatively slow pace.

An anaplastic astrocytoma is a grade III tumor. These rare tumorsrequire more aggressive treatment than benign pilocytic astrocytoma.

Astrocytoma Grade IV (also called Glioblastoma, previously named“Glioblastoma Multiforme,” “Grade IV Glioblastoma,” and “GBM”). Thereare two types of astrocytoma grade IV—primary, or de novo, andsecondary. Primary tumors are very aggressive and the most common formof astrocytoma grade IV. The secondary tumors are those which originateas a lower-grade tumor and evolve into a grade IV tumor.

Subependymal Giant Cell Astrocytoma—Subependymal giant cell astrocytomasare ventricular tumors associated with tuberous sclerosis.

Oligodendrogliomas can be found anywhere within the cerebral hemisphereof the brain, although the frontal and temporal lobes are the mostcommon locations. Sometimes oligodendrogliomas are mixed with other celltypes. These tumors may be graded using an “A to D” system, which isbased on microscopic features of the individual tumor cells. The gradeindicates how quickly the tumor cells reproduce and how aggressive thetumor is. About 4% of primary brain tumors are oligodendrogliomas,representing about 10-15% of the gliomas. Only 6% of these tumors arefound in infants and children. Most oligodendrogliomas occur in adultsages 50-60, and are found in men more often than women.

Mixed glioma (or oligoastrocytoma) usually contain a high proportion ofmore than one type of cell, most often astrocytes and oligodendrocytes.Occasionally, ependymal cells are also found. The behavior of a mixedglioma appears to depend on the grade of the tumor. It is less clearwhether their behavior is based on that of the most abundant cell type.

Ependymal cells line the ventricles of the brain and the center of thespinal cord. These tumors are divided into four major types:subependymomas (grade I), typically slow growing tumors; myxopapillaryependymomas (grade I), typically slow growing tumors; Ependymomas (gradeII), the most common of the ependymal tumors, which can be furtherdivided into the following subtypes, including cellular ependymomas,papillary ependymomas, clear cell ependymomas, and tancytic ependymomas;and anaplastic ependymomas (grade III), typically faster growing tumors.The various types of ependymomas appear in different locations withinthe brain and spinal column. Subependymomas usually appear near aventricle. Myxopapillary ependymomas tend to occur in the lower part ofthe spinal column. Ependymomas are usually located along, within, ornext to the ventricular system. Anaplastic ependymomas are most commonlyfound in the brain in adults and in the lower back part of the skull(posterior fossa) in children. They are rarely found in the spinal cord.Ependymomas are relatively rare tumors in adults, accounting for 2-3% ofprimary brain tumors. However, they are the sixth most common braintumor in children. About 30% of pediatric ependymomas are diagnosed inchildren younger than 3 years of age.

Optic gliomas may involve any part of the optic pathway, and they havethe potential to spread along these pathways. Most of these tumors occurin children under the age of 10. Grade I pilocytic astrocytoma and gradeII fibrillary astrocytoma are the most common tumors affecting thesestructures. Higher-grade tumors may also arise in this location. Twentypercent of children with neurofibromatosis (NF-1) will develop an opticglioma. These gliomas are typically grade I, pilocytic astrocytomas.Children with optic glioma are usually screened for NF-1 for thisreason. Adults with NF-1 typically do not develop optic gliomas.

Gliomatosis Cerebri is an uncommon brain tumor that is most frequentlypediatric and features widespread glial tumor cells in the brain. Thistumor is different from other gliomas because it is scattered andwidespread, typically involving two or more lobes of the brain. It couldbe considered a “widespread low-grade glioma” because it does not havethe malignant features seen in high-grade tumors.

Glioblastoma

Glioblastoma multiforme (GBM), a WHO grade IV malignant glioma,classification name “glioblastoma”, is the most common and mostaggressive primary brain tumor in adults. GBM accounts for 40%-60% ofall diffuse astrocytic tumors and 10%-15% of all intracranial neoplasticlesions. The biological characteristics of this tumor are exemplified byprominent proliferation, active invasiveness, and rich angiogenesis.(Nakada, M. et al., Cancers, 2011, 3: 3242-3278). GBM is composed ofpoorly differentiated neoplastic astrocytes. The presence ofmicrovascular proliferation and/or necrosis is essential forhistopathological diagnosis of GBM.

GBM is one of the most aggressive human cancers and is very difficult totreat due to several complicating factors: the tumor cells are veryresistant to conventional therapies; the brain is susceptible to damageby conventional therapy; the brain has a very limited capacity to repairitself; and many drugs cannot cross the blood-brain barrier to act onthe tumor.

Although treatment can involve radiation, surgery and chemotherapy withtemozolomide, which is an alkylating agent (P. J. Noughton et al. Clin.Cancer Res. (2000) 6:4110-4118), decades of surgical therapy,radiotherapy, and chemotherapy have failed to drastically changesurvival for GBM. The medium survival of patients with GBM in clinicaltrial populations treated with multimodal treatment approaches isapproximately 12-15 months, with only 3%-5% of patients surviving longerthan 36 months. (McNamara. M. G. et al., Cancers, 2013, 5: 1103-1119).

Glioblastoma multiforme has at least four distinct molecular subtypes.Tumor variants can be classified on the basis of somatic mutations inisocitrate dehydrogenase (IDH) ½ and TP53; transcriptional signature(classical, mesenchymal, neural or proneural), copy number variation,including co-deletion of chromosomes 1p and 19q; and amplification ormutation of the epidermal growth factor receptor (EGFR) and increasedDNA hypermethylation of promoter-associated CpG islands. (Parker, N. R.et al., “Molecular heterogeneity in glioblastoma: potential clinicalimplications,” Frontiers in Oncology 5, article 55 (March 2015)).

Classical GBM tumors are characterized by abnormally high levels ofepidermal growth factor receptor (EGFR) which is a protein found on thesurface of some cells that, when bound by epidermal growth factor, sendssignals for the cell to keep growing in number (proliferation). TheCancer Genome Atlas (TCGA) Research Network, Nature 455: 1061-1068(2008).

EGFR abnormalities occur at a much lower rate in the three other GBMsubtypes. The TP53 gene codes for tumor protein p53 that normallysuppresses tumor growth. TP53 is rarely mutated in classical GBM tumorssubtype, but is the most frequently mutated gene in other subtypes ofGBM.

Proneural GBM tumors are characterized by alterations of plateletderived growing factor receptor A (PDGFRA) and point mutations in IDH1,the gene that encodes isocitrate dehydrogenase 1. The gene IDH1, whenmutated, codes for a protein that can contribute to abnormal cellgrowth. PDGFRA, which plays an important role in cell proliferation,cell migration, and angiogenesis, was also found to be mutated andexpressed in abnormally high amounts. PDGFRA alteration only occurs inProneural tumors and not in any other subtypes. When PDGFRA is altered,too much of its protein can be produced, leading to uncontrolled tumorgrowth. The patients of this subtype tend to be younger and to survivelonger than in other subtypes.

The Mesenchymal subgroup contains the most frequent number of mutationsin the neurofibromatosis type 1 (NF1) tumor suppressor gene. Frequentmutations in the PTEN (phosphatase and tensin homolog) and TP53 tumorsuppressor genes also occur in the Mesenchymal subgroup. PTEN proteinacts as a tumor suppressor, helping regulate the cycle of cell division.

While the Neural subgroup has mutations in many of the same genes as theother groups, the group does not stand out from the others as havingsignificantly higher or lower rates of mutations. The Neural group ischaracterized by the expression of several markers that are also typicalof the brain's normal, noncancerous nerve cells, or neurons, such asNEFL, GABRA1, SYT1 and SLC12A5.

While classical and mesenchymal GBMs express gene expression profilesreminiscent of NSCs, IDH-mutant gliomas display a proneural phenotype.(Ilkanizadeh, et al., “Glial Progenitors as Targets for Transformationin Glioma,” Adv. Cancer Res. 121: 1-65 (2014)).

Based on clinical experience, two subgroups of otherwise histologicallyindistinguishable GBMs have been established: primary glioblastoma andsecondary glioblastoma. Primary glioblastoma, which comprises more than90% of biopsied or resected cases, arise de novo without antecedenthistory of low-grade disease, whereas secondary glioblastoma progressesfrom previously diagnosed low-grade gliomas. Primary glioblastomasdisplay classical mesenchymal and neural phenotypes, whereas secondaryglioblastomas tend to display a proneural phenotype that shifts toward amesenchymal phenotype with recurrence. (Parker, N. R. et al., “Molecularheterogeneity in glioblastoma: potential clinical implications,”Frontiers in Oncology 5, article 55 (March 2015)).

These molecular subtypes of glioblastoma multliforme appear to differ intheir clinical courses and therapeutic responses. For example, thedifferent subtypes show varying responses to aggressive chemotherapy andradiotherapy, with a difference of around 50% between the subtypes. Ithas been suggested that the pathology of each subtype might begin fromdifferent types of cells, which might explain the variation in responseto therapy. The greatest benefit was seen in the Classical andMesenchymal subtypes, where intensive therapy has significantly reducedmortality; and there was a suggestion of efficacy in the Neural subtype;but the Proneural subtype was less responsive to intensive therapyincluding conventional chemotherapy or chemo-radiation therapy.(Verhaak, R G, et al., Cancer Cell, 17(1):98-110, 2010))

Major Glioma Signaling Pathways

Several major signaling pathways have been associated with Glioma(Nakada, M. et al., Cancers, 2011, 3: 3242-3278).

1. Receptor Tyrosine Kinase Pathway (RTK/PI3K/Akt/mTOR Pathway).

The RTK/P13K/Akt pathway regulates various fundamental cellularprocesses such as proliferation, growth, apoptosis, and cytoskeletalrearrangement. The pathway involves receptor tyrosine kinases (RTKs),for example, epidermal growth factor receptor (EGFR), platelet derivedgrowing factor receptor (PDGFR), and vascular endothelial growth factorreceptor (VEGFR), etc., as well as tumor suppressor protein phosphatase,for example, phosphatase and tension homolog (PTEN), and protein kinasesPI3K, Akt, and mTOR. The Receptor Tyrosine Kinase pathway(RTK/PI3K/Akt/mTOR Pathway) is shown in FIG. 1.

EGFR gene amplification is the most frequent alteration (approximately40%) in GBM. EGFR is a transmembrane glycoprotein member of the ErbBreceptor family. In GBM, EGFR is dysregulated through overexpression,which arises because of EGFR gene amplification or activating mutationssuch as EGFRvIII that lead to ligand-independent signaling. EGFRaberrations have been correlated with a classical subtype of GBM (TCGAResearch Network, Nature 455: 1061-68; Verhaak, Roel G. W. et al.,Cancer Cell, 2010, 17: 98-110). Although it has been suggested thatalterations of EGFR may be correlated with increased aggressiveness ofGBM (Nakada, M. et al., Cancers, 2011, 3: 3242-3278), EGFR inhibitors(e.g., Gefinitib, Erlotinib) have not elicited clinical responses inpatients with GBMs in clinical trials (Rich, J. N., et al., N. Engl. J.Med. 2004, 351, 1260-1261; Haas-Kogan, D. A. et al., J. Natl. CancerInst. 2005, 97, 880-887; van den Bent, M. J. et al., J. Clin. Oncol.2009, 27, 1268-1274).

Overexpression of platelet-derived growth factor receptor (PDGFR),especially PDGFR-α and platelet-derived growth factor (PDGF) have beenobserved in astrocytic tumors of all grades, and their association withmalignant progression has been suggested (Nakada, M. et al., Cancers,2011, 3: 3242-3278). PDGFRA amplification (14%), as well as IDH1mutation, are major features of the Proneural subtype of GBM accordingto the TCGA classification (TCGA Research Network, Nature 455: 1061-68;Verhaak, R. G. et al., Cancer Cell, 2010, 17: 98-110). Despite deepassociation of this molecule with GBM, anti-PDGFR therapy using Imatinibyielded only limited clinical responses (Reardon, D. A., et al., J.Clin. Oncol. 2005, 23, 9359-9368; Reardon, D. A., et al., Br. J. Cancer2009, 101, 1995-2004).

The PI3Ks are widely expressed lipid kinases that promote diversebiological functions. The binding of PI3Ks and RTKs results inactivation of Akt through phosphatidylinositol 3,4,5-triphosphate (PiP3)and 3-phosphoinositide dependent protein kinase-1 (PDK1), which affectsmultiple fundamental cellular processes including cell survival,proliferation, and motility. According to the integrated genomicclassification of GBM, PI3K mutations (15%) are associated with theProneural subtype (TCGA Research Network, Nature, 2008, 455: 1061-68;Verhaak, R. G. et al., Cancer Cell, 2010, 17: 98-110).

Decreased PTEN activity can activate the RTKs/PI3K/Akt pathway sincePTEN negatively regulates the pathway by antagonizing PI3K function.Homozygous deletion or mutation of PTEN is a common genetic feature inGBM (40%), resulting in constitutive activation of the RTKs/PI3K/Aktpathway. PTEN loss is associated with both classical and mesenchymalsubtypes of GBM, according to the TCGA study (TCGA Research Network,Nature 455(23): 1061-68).

Akt is an STK (Serine/threonine specific protein kinase) that regulatescell growth, proliferation, and apoptosis. Akt activation has beenreported in approximately 80% of human GBMs and correlates with the factthat RTKs/PI3K/Akt signaling is altered in 88% of GBM (TCGA ResearchNetwork, Nature 455(23): 1061-68). Oncogenic Akt mutations have not beendetected in GBM. Akt inhibitor perifosine is undergoing clinicalevaluation in malignant gliomas (Nakada, M. et al., Cancers, 2011, 3:3242-3278).

2. p14ARF/MDM2/p53 Pathway

The p53 gene encodes a protein that responds to diverse cellularstresses to regulate target genes that induce cell cycle arrest, celldeath, cell differentiation, senescence, DNA repair, andneovascularization. Following DNA damage, p53 is activated and inducestranscription of genes (such as p21Waf1/Cip1) that function asregulators of cell cycle progression at G1 phase. Mouse double minute 2homolog (MDM2) oncogne inhibits p53 transcriptional activity by forminga tight complex with the p53 gene, and participates in the degradationof p53. The p14ARF gene codes a protein that directly binds to MDM2 andinhibits MDM2-mediated p53 degradation. In turn p14ARF expression isnegatively regulated by p53. Thus, inactivation of p14ARF/MDM2/p53 iscaused by altered expression of any of the p53, MDM2, or p14ARF genes.The p53 pathway plays a crucial role in the development of secondaryGBMs. The p53 gene is the most commonly mutated p53 pathway gene inglioma; however, molecular abnormalities involving other genes in thepathway have also been described. (Nakada, M. et al., Cancers, 2011, 3:3242-3278). The p14ARF/MDM2/p53 Pathway is shown in FIG. 2.

3. RB Pathway.

The RB (retinoblastoma tumor suppressor protein) pathway suppresses cellcycle entry and progression, as well as the p53 pathway. The 107-kDa RB1protein encoded by RB1 (at 13q14) controls progression through G1 intothe S-phase of the cell cycle (Serrano, M., et al., Nature, 1993, 366:704-707). The CDKN2A protein (i.e. p16INK4a which is cyclin-dependentkinase inhibitor 2A) binds to cyclin-dependent kinases 4 (CDK4) andinhibits the CDK4/cyclin D1 complex, thus inhibiting cell cycletransition from G1 to S phase. Thus, alteration of RB1, CDK4, or CDKN2Acan cause dysregulation of the G1-S phase transition. However,alteration of only the RB pathway is insufficient to induce tumorformation. EGFR amplification enhances the PI3K pro-growth pathway andis typically associated with CDKN2A deletions. CDKN2A loss is associatedwith the classical subtype of GBM, according to the TCGA study. (Nakada,M. et al., Cancers, 2011, 3: 3242-3278). The RB pathway is shown in FIG.3.

4. Ras/MEK/MAPK Pathway.

RAS (Rat sarcoma) proteins act as on/off (RAS-GDP/RAS-GTP) switchescontrolled by RTKs and neurofibromatosis type 1 tumor suppressor gene(NF-1). Activated RAS (RAS-GTP) then activates serine/threonine kinaseRAF. RAF activates mitogen-activated protein kinase kinase (MAPKK), alsocalled MEK, which in turn activates MAPK. MAPK activation results inactivation of various transcription factors, such as Elk1, c-myc, Ets,STAT1/3, and PPAR.

The NF-1 tumor suppressor gene encodes neurofibromin, which functionsprimarily as a RAS negative regulator and plays a role in adenylatecyclase- and Akt-mTOR-mediated pathways. There is increasing evidencethat the NF-1 gene is involved in the tumorigenesis of not onlyNF-1-related, but also sporadically occurring, gliomas. In the TCGApilot study, NF-1 mutation/homozygous deletions were identified in 18%of GBM. Mesenchymal GBMs, having frequent inactivation of the NF-1(37%), p53 (32%), and PTEN genes, respond to aggressive chemo-radiationadjuvant therapies. (Nakada, M. et al., Cancers, 2011, 3: 3242-3278).The Ras/MAPK pathway is shown in FIG. 4. A global view of the signalingpathways mentioned above is shown in FIG. 4.

Other signaling pathways may play a role in GBM initiation, migration,and invasion.

Brain Stem Cells

Glial cells outnumber neurons by 10-fold in the human brain and arecomposed mainly of terminally differentiated cells and minor discreteprecursor populations. (Ikanizadeh, S. et al., “Glial Progenitors astargets for transformation in glioma,” Adv. Cancer Res. 121: 1-65(2014)).

Two major germinal layers—the ventricular zone (VZ) and thesubventricular zone (SVZ)— give rise to most neurons and glial cells inthe forebrain. (Garcia-Verdugo, J M et al, “Architecture and Cell typesof the adlt subventricular zone: in search of the stem cells,” J.Neurobiol. 36: 234-48 (1998)). It was traditionally believed that thecapacity of these germinal layers to generate neurons was restricted tothe embryonic period; however, it is now known that new neurons continueto be added to certain regions of the adult vertebrate brain. In adultmammals, neuronal addition has been observed only in the olfactory bulband the hippocampus. New neurons destined for the olfactory bulb areborn in the SVZ of the lateral ventricles. A subpopulation of SVZ cellscan proliferate in culture, giving rise to spherical clusters of cells(neurospheres), which have the capacity to generate neurons, astrocytesand oligodendrocytes. Based on their ability to self-renew and theirpotential to give rise to multiple cell types, these SVZ-derived cellsare considered neural stem cells. Evidence also suggests that neuralstem cells (NSCs) line the third and fourth ventricles (Ikanizadeh, S.et al., “Glial Progenitors as targets for transformation in glioma,”Adv. Cancer Res. 121: 1-65 (2014), citing Weiss, S. et al, “MultipotentCNS stem cells are present in the adult mammalian spinal cord andventricular neuroaxis,” J. Neurosci. 16(23): 7599-7609 (1996); Xu, Y. etal., Neurogenesis in the ependymal layer of the adultrat 3^(rd)ventricle,” Exptl Neurol. 192(2): 251-64 (2005)).

Modeling of glioma in mice has shown that cells at variousdifferentiation stages throughout glial and neuronal lineages have thepotential to generate glioma. Recent advances highlight the cellularheterogeneity in gliomas, the influence of the tumor microenvironment,and that treatment-resistant tumor cells display a high degree ofstemness.

Transcriptomal profling of gliomas displaying a neuroepithelial origin,show that the mesenchymal phenotype is associated with stemness,invasiveness, and poor survival. Id. (citing H. S. Phillips, et al.,“Molecular subclasses of high-grade glioma predict prognosis, delineatea pattern of disease progression, and resemble stages in neurogenesis,”Cancer Cell. 9(3): 157-73 (2006); Sturm, D. et al, “Hotspot mutations inH3F3A and IDH1 define distinct epigenetic and biological subgroups ofglioblastoma,” Cancer Cell 22(4): 425-37 (2012); Verhaak, R G W, et al.,“Integrated genomic analysis identifies clinically relevant subtypes ofglioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, andNF1,” Cancer Cell. 17(1): 98-110 (2010)).

Cancer stem cells (CSCs) or tumor-initiating cells (TIC) are asubpopulation of tumor cells with the ability to undergo self-renewaland recapitulate the entire tumor population Wang, L. et al.,Interleukin-1β and transforming growth factor-β cooperate to induceneurosphere formation and increase tumorigenicity of adherent LN-229glioma cells,” Stem ell Res. & Therapy 3:5 (2012). Glioma stem cells(GSC) have been identified from human glioma tissues and glioma cellslines. Id. GSCs are characterized by the ability of self-renewal togenerate three-dimensional aggregates of cells in suspension termed“neurospheres” when cultured in serum-free conditions supplemented withepidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).Id. These glioma neurospheres reflect biological and pathologicalcharacteristics of primary gliomas, display resistance to chemo- andradio-therapies, and have enhanced oncogenic potential, generatingtumors that reproduce the characteristics of the original tumors afterintracranial transplantation. (Id., citing Ehrlicher, A. et al., Guidingneuronal growth with light,” Proc. Natl Acad. Sci. U.S.A. 99: 16024-8(2002); Difato, F. et al, “combined optical tweezers and laser dissectorfor controlled ablation of functional connections in neural networks,”J. Biomed. Opt. 16: 051306 (2011); Dictus, C. et al, “Comparativeanalysis of in vitro conditions for rat adult neural progenitor cells,”J. Neurosci Methods. 161: 250-58 (2007))

Animal Models of Glioma

The study of IDH-mutant gliomas has been obstructed by the lack ofmodels of IDH-mutant glioma-producing mice. Brain-specific IDH1^(R132H)knock-in mice are embryonically lethal. Izanizadeh, S. et al, citingSasaki, M. et al, “D-2 hydroxyglutarate produced by mutant IDH1 perturbscollagen maturation and basement membrane function,” Genes & Devel.26(18): 2038-49 (2012)). Cell lines with IDH1^(R132H) mutation can onlybe maintained transiently in vitro, since the mutation does not persistin non-immortalized cells. Primary IDH-mutant gliomas from patienttumors do not grow well in vitro (Id., citing Piaskowski, S. et al.,“Glioma acells showing IDH1 mutation cannot be propagated in standardcell culture conditions,” Br. J. Cancer 104(6): 968-70 (2011)). Incontrast to normal cells, introduction of IDH mutations into gliomacells decreases the proliferation rate, which may ultimately cause aselection pressure against cultured glioma cells harboring IDH mutations(Id., citing Bralten, L B C, et al., “IDH1 R132H decreases proliferationof glioma cell lines in vitro and in vivo,” Annals Neurol. 69(3): 455-63(2011)).

Spontaneous mouse models of GBM have been generated that are caused bymutation and therefore loss of three glioma relevant tumor suppressorgenes: Pten, p53 and NFL These mice have tumors that exhibithistopathological and molecular similarity with human GBM and haveprovided a powerful platform for natural history studies, molecularstudies and derivation of primary (Mut6) cells that can be maintained inlow passage culture and reintroduced in allografts to produce GBM(Llaguno S A et al., “Malignant Astrocytomas Originate from NeuralStem/Progenitor Cells in a Somatic Tumor Suppressor Mouse Model”, CancerCell. 2009 Jan. 6; 15(1): 45-56; Llaguno S A et al., “Neural and CancerStem Cells in Tumor Suppressor Mouse Models of Malignant Astrocytoma”,Cold Spring Harb Symp Quant Biol. 2008; 73: 421-426).

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

Y¹ is selected from the group consisting of C═O, CH₂, and SO₂;

n=1, 2, or 3;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, NR₂. In the context of this paragraph, R is selected fromthe group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, Et; and

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I-a:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

n=1, 2, or 3;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et; and

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, n=1 or 2; R³═H; and R⁴ is selected from thegroup consisting of H, Me, and propargyl. According to anotherembodiment, R¹ and R² are independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, andacyloxyalkyl. According to another embodiment, X¹ is selected from thegroup consisting of H, F, Cl, CN, NH₂, NO₂, N₃, and SO₂Me; and X²=L²-R⁶.

According to another aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I-b:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et; and

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, n=1 or 2; R¹ and R² are independentlyselected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆ alkynyl,C₁-C₆ hydroxyalkyl, and acyloxyalkyl; R³═H; and R⁴ is selected from thegroup consisting of H, Me, and propargyl.

According to another aspect, the described invention provides a smallmolecule anti-proliferative compound of Formula I-c:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y² is selected from the group consisting of CR′R″, NR, O, and S. In thecontext of this paragraph, R, R′, and R″ are independently selected fromthe group consisting of H, F, Me, Et, i-Pr, and cyclopropyl;

k=0, 1, 2, or 3;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl; and

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, Y² is selected from the group consisting ofCH₂, NR, O, and S. In the context of this paragraph, R is selected fromthe group consisting of H and Me; k=1 or 2; n=1 or 2; L² is selectedfrom the group consisting of NH, O, S, CHOH, C═O, —S(CH₂)—; and R³═H;and R⁴ is selected from the group consisting of H, Me, and propargyl.According to another embodiment, L² is selected from the groupconsisting of NH, O, and S; and R⁶ is selected from the group consistingof aryl, and heteroaryl.

According to another aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I-d:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y³ is selected from the group consisting of CH₂, NR, O, and S. In thecontext of this paragraph, R is selected from the group consisting of H,Me, CD₃, CF₃, Et, isopropyl, and cyclopropyl.

n=1 or 2;

L² is selected from the group consisting of S, O, and NH;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, andC₂-C₆ alkynyl; and

R⁶ is selected from the group consisting of aryl, heteroaryl, benzyl,fused heteroarylaryl, fused arylheteroaryl, and fused arylaryl;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, Y³ is selected from the group consisting ofCH₂, NH, NMe, and O; R⁴ is selected from the group consisting of H, Me,and propargyl; and R⁶ is selected from the group consisting of aryl andheteroaryl.

According to another aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I-e:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y⁴ is selected from the group consisting of CR′R″, NR, O, and S. In thecontext of this paragraph, R, R′, and R″ are independently selected fromthe group consisting of H, F, Me, Et, isopropyl and cyclopropyl;

j=0, 1, 2, or 3;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl; and

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, Y⁴═CH₂; j=1; n=1 or 2; L² is selected fromthe group consisting of S, O, NH, CHOH, C═O, and —S(CH₂)—; R² isselected from the group consisting of H, C₁-C₃ alkyl, propargyl, C₁-C₃hydroxyalkyl, and acyloxyalkyl; and R⁴ is selected from the groupconsisting of H, Me, and propargyl.

According to another aspect, the described invention provides a smallmolecule anti-cancer compound of Formula I-f:

wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et; and

R¹⁴ and R¹⁵ can be attached at any available position on the aromaticring and are selected from the group consisting of H, D, F, Cl, Br, CF₃,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, cycloalkyl, OR, NR₂, NO₂, N₃,CN, CO₂R, CO₂NR₂, SR, alkylacyl and arylacyl. In the context of thisparagraph, R is independently selected from the group consisting of H,Me, Et, isopropyl, cyclopropyl, propargyl, and acyl;

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, n=1 or 2; L² is selected from the groupconsisting of NH, O, S, —S(CH₂)—, C═O, and CHOH; R¹ and R² areindependently selected from the group consisting of H, C₁-C₃ alkyl,propargyl, C₁-C₃ hydroxyalkyl, and acyloxyalkyl; R¹ and R² mayoptionally form a ring such that R¹-R² consists of a four or fivesubunit chain comprising subunits independently selected from the groupconsisting of CH₂, NH, NMe, and O; R¹ and R³ may optionally form a ringsuch that R¹-R³═—(CH₂)₃—; R² and R⁴ may optionally form a ring such thatR²-R⁴ consists of a two subunit chain comprising subunits independentlyselected from the group consisting of CH₂ and CH-alkyl, and R² maysimultaneously form a ring with R¹ as described above; R³═H; R⁴ isselected from the group consisting of H, Me, and propargyl; and R¹⁴ andR¹⁵ can be attached at any available position on the aromatic ring andare independently selected from the group consisting of H, F, Cl, Br,CF₃, C₁-C₃ alkyl, propargyl, OR, NR₂, NO₂, CN, CO₂R, and CO₂NR₂, SR. Inthe context of this paragraph, R is independently selected from thegroup consisting of H, Me, Et and propargyl.

According to another aspect, the described invention provides a compoundselected from the group consisting of:

According to one embodiment, the compound is in form of a pharmaceuticalcomposition comprising a therapeutic amount of the compound and apharmaceutically acceptable carrier.

According to another aspect, the described invention provides a methodof treating a cancer in a subject in need thereof, comprisingadministering to the subject the pharmaceutical composition of thedescribed invention, wherein the therapeutic amount is effective toinhibit tumor growth, inhibit tumor proliferation, induce cell death, ora combination thereof.

According to one embodiment, the therapeutic amount is effective toinhibit a cholesterol biosynthesis pathway. According to anotherembodiment, the therapeutic amount is effective to down-regulate SHREBP2and its target genes. According to another embodiment, the cancer is asolid brain tumor. According to another embodiment, the solid braintumor is a glioma. According to another embodiment, the glioma is aglioblastoma. According to another embodiment, the solid brain tumorcomprises cancer stem cells. According to another embodiment, thetherapeutic amount of the composition is effective to selectivelyinhibit growth of cancer cells, proliferation of cancer cells, to inducecell death of cancer cells, or a combination thereof, without affectingnormally dividing cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the Receptor Tyrosine Kinase (RTK/PI3K/Akt/mTOR) pathway.

FIG. 2 depicts the p14ARF/MDM2/p53 pathway.

FIG. 3 depicts the retinoblastoma (RB) tumor suppressor protein pathway

FIG. 4 depicts the Ras/MEK/MAPK pathway.

FIG. 5 depicts a global view of the Receptor Tyrosine Kinase(RTK/PI3K/Akt/mTOR) pathway; the p14ARF/MDM2/p53 pathway; theretinoblastoma (RB) tumor suppressor protein pathway and theRas/MEK/MAPK pathway.

FIG. 6 comprising FIG. 6(a), FIG. 6(b), FIG. 6(c) and FIG. 6(d) showsthe overall screening strategy against glioblastoma tumor derivedneuronal stem cells Mut6. FIG. 6(a) is a schematic showing that primaryneuronal stem cells isolated from mouse GBM tumors (Mut6; glioblastoma)were used in a luminescence-based, cell viability high throughputprimary screening assay against ˜200,000 synthetic compounds, whichyielded 4,480 positive hits with >50% inhibition of cell ATPconsumption. The 4,480 compounds were further screened to identify 1,078compounds that displayed greater than or equal to 70% inhibition of Mut6ATP consumption. The 1,078 compounds were screened for toxicity againstnormally dividing wild-type mouse embryonic fibroblasts (MEFs),wild-type mouse astrocytes and wild-type mouse SVZ stem cells; andcompounds toxic to all three cell-types were removed, which resulted inthe identification of 713 compounds. FIG. 6(b) shows the results fromthe toxicity screen which identified 713 compounds. FIG. 6(c) showscounterscreeing of the 713 compounds against normal astrocytes and mouseembryonic fibroblasts (MEFs) which identified 61 compounds that onlykill cancer stem cells. FIG. 6(d) is a schematic showing that the 61compounds were analyzed by S9 fraction assay and hepatocyte assay whichidentified 17 candidate compounds.

FIG. 7 comprising FIG. 7A, FIG. 7B and FIG. 7C shows that compound 4C12induces cell death in Mut6 tumor cells. Normal astrocytes, Mut6 cellsand mouse embryonic fibroblasts were treated with increasingconcentrations of compound 4C12. FIG. 7A is a plot of relative ATPactivity (y axis, a measure of viability) versus concentration (nM),which shows that compound 4C12 has an ED50 of 50 nM against Mut6 cells.Normal astrocytes and mouse embryonic fibroblasts (MEFs) were unaffectedby compound 4C12. FIG. 7B shows phase contrast microscopy of Mut6 cellsand control MEFs after 14 hours, 23 hours and 38 hours treatment withcompound 4C12 and a negative control containing vehicle only (Ctrl).FIG. 7C is a plot of live cells (%) versus time (hrs) which shows thatall cells treated with vehicle (negative control (Ctrl)) remainedviable, while only 50% of the Mut6 tumor cells treated with compound4C12 were viable after 96 hours.

FIG. 8 comprising FIG. 8A and FIG. 8B shows the identification ofmolecular changes between 6 hr and 24 hr time points. FIG. 8A shows aschematic of a cell-based assay. Cells were plated 48 hours beforetreatment with test compounds Compound 1, Compound 2 and 4C12, and anegative control. Cultures then were treated with the test compound. Themedia was removed and cells washed twice. Fresh media was added withoutcompound. Data points were 6 hr, 22 hr, 33 hr, 51 hr, 71 hr andcontinuous. Molecular changes were identified between the 6 hr and 24 hrtime points. An ATP assay was performed at 96 hours. On day 2, Cellstreated with 4C12 were round-shaped. On day 3, the cells were arrestedin G2. On day 4, 10-20% of the cells were apoptotic. By day 5, 80-90% ofthe cells were apoptotic. FIG. 8B shows a plot of relative ATP activity(y axis) versus time (x axis) showing that cells treated with compoundsCompound 1, Compound 2 and 4C12 reduced ATP activity as a function oftime relative to the control (Ctrl).

FIG. 9 comprising figures FIG. 9A to FIG. 9QQQ shows 1050 curves foreach analog compound in Table A plotted as ATP activity (y axis, ameasure of viability) vs. concentration (nM) (x-axis). FIG. 9A shows thechemical structure and 1050 curve of analog compound DS-1-033; FIG. 9Bshows the chemical structure and 1050 curve of analog compound DS-1-023;FIG. 9C shows the chemical structure and 1050 curve of analog compoundDS-1-031. FIG. 9D shows the chemical structure and 1050 curve of analogcompound DS-1-043; FIG. 9E shows the chemical structure and 1050 curveof analog compound DS-1-053; FIG. 9F shows the chemical structure and1050 curve of analog compound DS-1-055; FIG. 9G shows the chemicalstructure and 1050 curve of analog compound DS-1-061; FIG. 9H shows thechemical structure and 1050 curve of analog compound DS-1-063; FIG. 9Ishows the chemical structure and 1050 curve of analog compound DS-1-065;FIG. 9J shows the chemical structure and 1050 curve of analog compoundDS-1-067; FIG. 9K shows the chemical structure and 1050 curve of analogcompound DS-1-069; FIG. 9L shows the chemical structure and 1050 curveof analog compound DS-1-071; FIG. 9M shows the chemical structure and1050 curve of analog compound DS-1-075; FIG. 9N shows the chemicalstructure and 1050 curve of analog compound DS-1-077; FIG. 9O shows thechemical structure and 1050 curve of analog compound DS-1-079; FIG. 9Pshows the chemical structure and 1050 curve of analog compound DS-1-085;FIG. 9Q shows the chemical structure and 1050 curve of analog compoundDS-1-089; FIG. 9R shows the chemical structure and 1050 curve of analogcompound DS-1-103; FIG. 9S shows the chemical structure and 1050 curveof analog compound DS-1-105; FIG. 9T shows the chemical structure and1050 curve of analog compound DS-1-117; FIG. 9U shows the chemicalstructure and 1050 curve of analog compound DS-1-119; FIG. 9V shows thechemical structure and 1050 curve of analog compound DS-1-123; FIG. 9Wshows the chemical structure and 1050 curve of analog compound DS-1-125;FIG. 9X shows the chemical structure and 1050 curve of analog compoundDS-1-129; FIG. 9Y shows the chemical structure and 1050 curve of analogcompound DS-1-131; FIG. 9Z shows the chemical structure and 1050 curveof analog compound DS-1-133; FIG. 9AA shows the chemical structure and1050 curve of analog compound DS-1-135; FIG. 9BB shows the chemicalstructure and 1050 curve of analog compound DS-1-137; FIG. 9CC shows thechemical structure and 1050 curve of analog compound DS-1-139; FIG. 9DDshows the chemical structure and 1050 curve of analog compound DS-1-163;FIG. 9EE shows the chemical structure and 1050 curve of analog compoundDS-1-177; FIG. 9FF shows the chemical structure and 1050 curve of analogcompound DS-1-179; FIG. 9GG shows the chemical structure and 1050 curveof analog compound DS-1-181; FIG. 9HH shows the chemical structure and1050 curve of analog compound DS-1-183; FIG. 9II shows the chemicalstructure and 1050 curve of analog compound DS-1-185; FIG. 9JJ shows thechemical structure and 1050 curve of analog compound DS-1-191; FIG. 9KKshows the chemical structure and 1050 curve of analog compound DS-1-195;FIG. 9LL shows the chemical structure and 1050 curve of analog compoundDS-1-209; FIG. 9MM shows the chemical structure and 1050 curve of analogcompound DS-1-205 (biotin); FIG. 9NN shows the chemical structure and1050 curve of analog compound DS-1-213; FIG. 9OO shows the chemicalstructure and 1050 curve of analog compound DS-1-217; FIG. 9PP shows thechemical structure and 1050 curve of analog compound DS-1-225; FIG. 9QQshows the chemical structure and 1050 curve of analog compound DS-1-227;FIG. 9RR shows the chemical structure and 1050 curve of analog compoundDS-1-231; FIG. 9SS shows the chemical structure and 1050 curve of analogcompound DS-1-239; FIG. 9TT shows the chemical structure and 1050 curveof analog compound DS-1-241; FIG. 9UU shows the chemical structure and1050 curve of analog compound DS-1-261; FIG. 9VV shows the chemicalstructure and 1050 curve of analog compound DS-1-192; FIG. 9WW shows thechemical structure and 1050 curve of analog compound DS-1-265; FIG. 9XXshows the chemical structure and 1050 curve of analog compound DS-1-269;FIG. 9YY shows the chemical structure and 1050 curve of analog compoundDS-1-271; FIG. 9ZZ shows the chemical structure and 1050 curve of analogcompound DS-1-275; FIG. 9AAA shows the chemical structure and 1050 curveof analog compound DS-1-279; FIG. 9BBB shows the chemical structure and1050 curve of analog compound DS-1-283; FIG. 9CCC shows the chemicalstructure and 1050 curve of analog compound DS-1-287; FIG. 9DDD showsthe chemical structure and 1050 curve of analog compound DS-1-291; FIG.9EEE shows the chemical structure and 1050 curve of analog compoundDS-1-295; FIG. 9FFF shows the chemical structure and 1050 curve ofanalog compound DS-1-299; FIG. 9GGG shows the chemical structure and1050 curve of analog compound DS-1-301; FIG. 9HHH shows the chemicalstructure and 1050 curve of analog compound DS-1-305; FIG. 9III showsthe chemical structure and 1050 curve of analog compound DS-2-035; FIG.9JJJ shows the chemical structure and 1050 curve of analog compoundDS-2-045; FIG. 9KKK shows the 1050 curve of analog compoundDS-2-045-072214; FIG. 9LLL shows the chemical structure and 1050 curveof analog compound DS-2-051; FIG. 9MMM shows the chemical structure and1050 curve of analog compound DS-2-057; FIG. 9NNN shows the chemicalstructure and 1050 curve of analog compound JCH-109; FIG. 9OOO shows thechemical structure and 1050 curve of analog compound DS-2-053; FIG. 9PPPshows the chemical structure and 1050 curve of analog compound DS-2-055;FIG. 9QQQ shows the 1050 curve of analog compound DS-2-045-072214.

FIG. 10 shows a table depicting the results of microarray analysis toidentify over-represented pathways associated with treatment withcompound 4C12 for 48 hours.

FIG. 11 is an illustration showing that cholesterol homeostasis in atypical mammalian cell is achieved via at least four major routes (takenfrom Jiang W and Song, B L, “Ubiquitin ligases in cholesterolmetabolism,” Diabetes Metab. J. 38: 171-180 (2014).

FIG. 12 comprising FIG. 12A and FIG. 12B shows that cholesterol levelsare decreased, TG levels are increased and cholesterol synthesis enzymesare down-regulated by 4C12. FIG. 12A shows a bar graphs showingcholesterol (pg/cell) (left graph), and triglyceride (pg/cell) (rightgraph) for Mut6 cells treated with a negative control (vehicle only(ctrl)), and with compound 4C12 for 24 hours and 48 hours. The figuresshow that in the presence of compound 4C12, cholesterol level decreases,and triglyceride level increases. FIG. 12B shows a bar graph of relativemRNA vs. enzymes of cholesterol synthesis showing that genes forcholesterol synthesis enzymes are down-regulated by compound 4C12. Mut6cells were treated with compound 4C12 for 24 hours and then mRNA levelsfor Hydroxymethylglutaryl-CoA synthase(Hmgcs);3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr); acetoacetyl-CoAsynthetase (AACS); Delta(24)-sterol reductase (Dhcr24);7-dehydrocholesterol reductase (Dhcr7), Sterol C5-desaturase (Sc5d);Squalene synthase (SS); and farnesyl pyrophosphate (FPP) synthase (FPPS)were determined.

FIG. 13 comprising FIG. 13(a), FIG. 13(b), FIG. 13(c), FIG. 13(d), FIG.13(e) and FIG. 13(f) shows that 4C12 inhibits Srebp2 activity. FIG.13(a) is a bar graph showing cholesterol gene profile for Mut6 cellswere treated with compound 4C12 for 2 hr compared to a DMSO control.FIG. 13(b) is a bar graph showing cholesterol gene profile for Mut6cells were treated with compound 4C12 for 9 hr compared to a DMSOcontrol. FIG. 13(c) is a bar graph showing cholesterol gene profile forMut6 cells were treated with compound 4C12 for 16 hr compared to a DMSOcontrol. FIG. 13(d) is a bar graph showing cholesterol gene profile forMut6 cells were treated with compound 4C12 for 24 hr compared to a DMSOcontrol. FIG. 13(e) is a bar graph showing cholesterol gene profile forMut6 cells were treated with compound 4C12 for 48 hr compared to a DMSOcontrol. The data shows that compound 4C12 inhibits Srebp2 target genes,and not Srebp1 target genes. FIG. 13(f) shows a Western blot. Mut6 cellswere treated with vehicle only (−) or with compound 4C12 (+) for 7 hr(left panel), 13 hr (middle panel) and 23 hr (right panel). Cells werecollected, lysed in SDS buffer, subjected to SDS PAGE, and cell proteinstransferred to a membrane by a standard protocol. The membrane waswashed, treated with antibodies to SREBP1, SREBP2 and a positive control(Cadherin), rewashed and bound antibodies then revealed. The blotsshowed that compound 4C12 was effective to decrease SREBP2 protein.

FIG. 14 comprising FIG. 14A and FIG. 14B shows that Compactin (statin)and 4C12 make a combination effect. FIG. 14A is a bar graph plotting ATPactivity (a measure of viability) for Mut6 cells treated withcompactin/mevastatin, compound 4C12, and the combination ofcompactin/mevastatin+compound 4C12, versus a negative control (vehicleonly (Ctrl)). The results show that the combination ofcompactin/mevastatin and compound 4C12 exert an effect greater than eachdoes alone. FIG. 14B illustrates a statin's inhibitory effect on themevalonate arm of the cholesterol biosynthesis pathway.

FIG. 15 comprising FIG. 15(a), FIG. 15(b), FIG. 15(c) and FIG. 15(d)shows that Compactin (statin) and 4C12 make a combination effect; thataddition of cholesterol inhibits 4C12-induced cell-death; and activationof SREBP2 by knock-down of Insig1/Insig2 makes cells less sensitive to4C12. FIG. 15(a) is a bar graph plotting ATP activity (a measure ofviability) for Mut6 cells treated with compactin/mevastatin, compound4C12, and the combination of compactin/mevastatin+compound 4C12, versusa negative control (vehicle only (Ctrl)). FIG. 15 (b) is a bar graphshowing ATP activity (a measure of viability) vs. concentration ofcholesterol (μM)—Mut6 cells were treated with compound 4C12 versus acells treated with DMSO (negative control). Addition of cholesterolinhibits compound 4C12-induced cell-death. FIG. 15(c) shows a graph ofrelative ATP activity (y-axis, a measure of viability) vs. compound 4C12concentration (nM (x axis)). Addition of SREBP2 by knock-down ofInsig1/Insig2 makes Mut6 cells less sensitive to compound 4C12. FIG.15(d) illustrates that Insig acts to suppress Srebp2 activity.

FIG. 16 comprising FIG. 16A and FIG. 16B shows that Srebp2 target geneswere not decreased by 4C12 in MEFs and astrocytes. FIG. 16A is a bargraph of relative mRNA level for cholesterol biosynthesis pathway targetgenes Hmgcs, Hmgcr, FPPS, LDLR; and SREBP1c in mouse embryonicfibroblasts (MEFs) treated with compound 4C12 or dmso (negativecontrol). FIG. 16B is a bar graph of relative mRNA level for cholesterolbiosynthesis pathway target genes Hmgcs, Hmgcr, FPPS, LDLR; and SREBP1cin astrocytes treated with compound 4C12 or dmso (negative control). Thefigure shows that Srebp2 target genes were not decreased by compound4C12 in MEFs and Astrocytes.

FIG. 17 comprising FIG. 17A and FIG. 17B compares cholesterol level(pg/cell) and triglyceride level (pg/cell) in Mut6 cells and in mouseembryonic fibroblast (MEF) cells treated with compound 4C12 for 24 hoursand 48 hours to a negative control (vehicle). FIG. 17A shows cholesterollevel (pg/cell) in Mut6 cells and in mouse embryonic fibroblast (MEF)cells treated with compound 4C12 for 24 hours and 48 hours to a negativecontrol (vehicle). FIG. 17B shows triglyceride level (pg/cell) in Mut6cells and in mouse embryonic fibroblast (MEF) cells treated withcompound 4C12 for 24 hours and 48 hours to a negative control (vehicle).The results show that the observed decrease in cholesterol by compound4C12 is specific to Mut6 tumor cells, and that Mut6 cells have a muchlower basal level of cholesterol and triglycerides.

FIG. 18 comprising FIG. 18A and FIG. 18B shows the effect of inhibitionof cholesterol biosynthesis pathway genes Hmgcs and the effect ofinhibition of ABCa1. FIG. 18A is a bar graph showing the effect ofinhibition of cholesterol biosynthesis pathway genes Hmgcs; Hmgcr, AACS,Dhcr24, Dhcr7, Sc5d, SS, FPPS, LDLR; and SREBP2 in Mut6 cells treatedwith DMSO or with compound 4C12 for 16 hours. FIG. 18B is a bar graphshowing effect on level of ABCa1 of treating Mut6 cells with compound4C12 for 16 hours versus a DMSO negative control.

FIG. 19 comprising FIG. 19A, FIG. 19B, FIG. 19C and FIG. 19D showsrelative ATP level (y-axis, a measure of viability) vs. concentration ofcompound 4C12 (nM). FIG. 19A shows relative ATP level (y-axis, a measureof viability) vs. concentration of compound 4C12 (nM) for variousprimary patient derived glioblastoma cell lines. FIG. 19B shows relativeATP level (y-axis, a measure of viability) vs. concentration of compound4C12 (nM) for various primary patient derived glioblastoma cell lines.FIG. 19C shows relative ATP level (y-axis, a measure of viability) vs.concentration of compound 4C12 (nM) for HeLa (cervical), HT-29 (colon),435 (breast), 549 (lung), MCF7 (breast), HCC38 (breast), Daoy(medulloblastoma (brain) cancer cell lines, and mouse embryonicfibroblast (MEF) cells in the presence of serum. FIG. 19D shows relativeATP level (y-axis, a measure of viability) vs. concentration of compound4C12 (nM) for HeLa (cervical), HT-29 (colon), 435 (breast), 549 (lung),MCF7 (breast), HCC38 (breast), Daoy (medulloblastoma (brain) cancer celllines, and mouse embryonic fibroblast (MEF) cells in the absence ofserum.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Various terms used throughout this specification shall have thedefinitions set out herein.

The term “adjuvant therapy” refers to a treatment added to a primarytreatment to prevent recurrence of a disease, or the additional therapygiven to enhance or extend the primary therapy's effect, as inchemotherapy's addition to a surgical regimen.

The term “agonist” as used herein refers to a chemical substance capableof activating a receptor to induce a full or partial pharmacologicalresponse. Receptors can be activated or inactivated by either endogenousor exogenous agonists and antagonists, resulting in stimulating orinhibiting a biological response. A physiological agonist is a substancethat creates the same bodily responses, but does not bind to the samereceptor. An endogenous agonist for a particular receptor is a compoundnaturally produced by the body which binds to and activates thatreceptor. A superagonist is a compound that is capable of producing agreater maximal response than the endogenous agonist for the targetreceptor, and thus an efficiency greater than 100%. This does notnecessarily mean that it is more potent than the endogenous agonist, butis rather a comparison of the maximum possible response that can beproduced inside a cell following receptor binding. Full agonists bindand activate a receptor, displaying full efficacy at that receptor.Partial agonists also bind and activate a given receptor, but have onlypartial efficacy at the receptor relative to a full agonist. An inverseagonist is an agent which binds to the same receptor binding-site as anagonist for that receptor and reverses constitutive activity ofreceptors. Inverse agonists exert the opposite pharmacological effect ofa receptor agonist. An irreversible agonist is a type of agonist thatbinds permanently to a receptor in such a manner that the receptor ispermanently activated. It is distinct from a mere agonist in that theassociation of an agonist to a receptor is reversible, whereas thebinding of an irreversible agonist to a receptor is believed to beirreversible. This causes the compound to produce a brief burst ofagonist activity, followed by desensitization and internalization of thereceptor, which with long-term treatment produces an effect more like anantagonist. A selective agonist is specific for one certain type ofreceptor.

The term “antagonist” as used herein refers to a small molecule,peptide, protein, or antibody that can bind to an enzyme, a receptor ora co-receptor, competitively or noncompetitively through a covalentbond, ionic bond, hydrogen bond, hydrophobic interaction, or acombination thereof and either directly or indirectly deactivate arelated downstream signaling pathway.

The term “anti-cancer compounds” as used herein refers to small moleculecompounds that selectively target cancer cells and reduce their growth,proliferation, or invasiveness, or tumor burden of a tumor containingsuch cancer cells

The term “administering” as used herein includes in vivo administration,as well as administration directly to tissue ex vivo. Generally,compositions may be administered systemically either orally, buccally,parenterally, topically, by inhalation or insufflation (i.e., throughthe mouth or through the nose), or rectally in dosage unit formulationscontaining conventional nontoxic pharmaceutically acceptable carriers,adjuvants, and vehicles as desired, or may be administered by means suchas, but not limited to, injection, implantation, grafting, topicalapplication, or parenterally.

The terms “analog” and “derivative” are used interchangeably to mean acompound produced from another compound of similar structure in one ormore steps. A “derivative” or “analog” of a compound retains at least adegree of the desired function of the reference compound. Accordingly,an alternate term for “derivative” may be “functional derivative.”Derivatives can include chemical modifications, such as akylation,acylation, carbamylation, iodination or any modification thatderivatizes the compound. Such derivatized molecules include, forexample, those molecules in which free amino groups have beenderivatized to form amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformal groups. Free carboxyl groups can be derivatized to form salts,esters, amides, or hydrazides. Free hydroxyl groups can be derivatizedto form O-acyl or O-alkyl derivatives.

The term “allosteric modulation” as used herein refers to the process ofmodulating a receptor by the binding of allosteric modulators at adifferent site (i.e., regulatory site) other than of the endogenousligand (orthosteric ligand) of the receptor and enhancing or inhibitingthe effects of the endogenous ligand. It normally acts by causing aconformational change in a receptor molecule, which results in a changein the binding affinity of the ligand. Thus, an allosteric ligand“modulates” its activation by a primary “ligand” and can adjust theintensity of the receptor's activation. Many allosteric enzymes areregulated by their substrate, such a substrate is considered a“homotropic allosteric modulator.” Non-substrate regulatory moleculesare called “heterotropic allosteric modulators.”

The term “allosteric regulation” is the regulation of an enzyme or otherprotein by binding an effector molecule at the proteins allosteric site(meaning a site other than the protein's active site). Effectors thatenhance the protein's activity are referred to as “allostericactivators”, whereas those that decrease the protein's activity arecalled “allosteric inhibitors.” Thus, “allosteric activation” occurswhen the binding of one ligand enhances the attraction between substratemolecules and other binding sites; “allosteric inhibition” occurs whenthe binding of one ligand decrease the affinity for substrate at otheractive sites. The term “antagonist” as used herein refers to a substancethat counteracts the effects of another substance.

The terms “apoptosis” or “programmed cell death” refer to a highlyregulated and active process that contributes to biologic homeostasiscomprising a series of biochemical events that lead to a variety ofmorphological changes, including blebbing, changes to the cell membrane,such as loss of membrane asymmetry and attachment, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomal DNAfragmentation, without damaging the organism.

Apoptotic cell death is induced by many different factors and involvesnumerous signaling pathways, some dependent on caspase proteases (aclass of cysteine proteases) and others that are caspase independent. Itcan be triggered by many different cellular stimuli, including cellsurface receptors, mitochondrial response to stress, and cytotoxic Tcells, resulting in activation of apoptotic signaling pathways

The caspases involved in apoptosis convey the apoptotic signal in aproteolytic cascade, with caspases cleaving and activating othercaspases that then degrade other cellular targets that lead to celldeath. The caspases at the upper end of the cascade include caspase-8and caspase-9. Caspase-8 is the initial caspase involved in response toreceptors with a death domain (DD) like Fas.

Receptors in the TNF receptor family are associated with the inductionof apoptosis, as well as inflammatory signaling. The Fas receptor (CD95)mediates apoptotic signaling by Fas-ligand expressed on the surface ofother cells. The Fas-FasL interaction plays an important role in theimmune system and lack of this system leads to autoimmunity, indicatingthat Fas-mediated apoptosis removes self-reactive lymphocytes. Fassignaling also is involved in immune surveillance to remove transformedcells and virus infected cells. Binding of Fas to oligimerized FasL onanother cell activates apoptotic signaling through a cytoplasmic domaintermed the death domain (DD) that interacts with signaling adaptorsincluding FAF, FADD and DAX to activate the caspase proteolytic cascade.Caspase-8 and caspase-10 first are activated to then cleave and activatedownstream caspases and a variety of cellular substrates that lead tocell death.

Mitochondria participate in apoptotic signaling pathways through therelease of mitochondrial proteins into the cytoplasm. Cytochrome c, akey protein in electron transport, is released from mitochondria inresponse to apoptotic signals, and activates Apaf-1, a protease releasedfrom mitochondria. Activated Apaf-1 activates caspase-9 and the rest ofthe caspase pathway. Smac/DIABLO is released from mitochondria andinhibits IAP proteins that normally interact with caspase-9 to inhibitapoptosis. Apoptosis regulation by Bcl-2 family proteins occurs asfamily members form complexes that enter the mitochondrial membrane,regulating the release of cytochrome c and other proteins. TNF familyreceptors that cause apoptosis directly activate the caspase cascade,but can also activate Bid, a Bcl-2 family member, which activatesmitochondria-mediated apoptosis. Bax, another Bcl-2 family member, isactivated by this pathway to localize to the mitochondrial membrane andincrease its permeability, releasing cytochrome c and othermitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation,blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor)is a protein found in mitochondria that is released from mitochondria byapoptotic stimuli. While cytochrome C is linked to caspase-dependentapoptotic signaling, AIF release stimulates caspase-independentapoptosis, moving into the nucleus where it binds DNA. DNA binding byAIF stimulates chromatin condensation, and DNA fragmentation, perhapsthrough recruitment of nucleases.

The mitochondrial stress pathway begins with the release of cytochrome cfrom mitochondria, which then interacts with Apaf-1, causingself-cleavage and activation of caspase-9. Caspase-3, -6 and -7 aredownstream caspases that are activated by the upstream proteases and actthemselves to cleave cellular targets.

Granzyme B and perforin proteins released by cytotoxic T cells induceapoptosis in target cells, forming transmembrane pores, and triggeringapoptosis, perhaps through cleavage of caspases, althoughcaspase-independent mechanisms of Granzyme B mediated apoptosis havebeen suggested.

Fragmentation of the nuclear genome by multiple nucleases activated byapoptotic signaling pathways to create a nucleosomal ladder is acellular response characteristic of apoptosis. One nuclease involved inapoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse(CAD). DFF/CAD is activated through cleavage of its associated inhibitorICAD by caspases proteases during apoptosis. DFF/CAD interacts withchromatin components such as topoisomerase II and histone H1 to condensechromatin structure and perhaps recruit CAD to chromatin. Anotherapoptosis activated protease is endonuclease G (EndoG). EndoG is encodedin the nuclear genome but is localized to mitochondria in normal cells.EndoG may play a role in the replication of the mitochondrial genome, aswell as in apoptosis. Apoptotic signaling causes the release of EndoGfrom mitochondria. The EndoG and DFF/CAD pathways are independent sincethe EndoG pathway still occurs in cells lacking DFF.

Hypoxia, as well as hypoxia followed by reoxygenation can triggercytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) aserine-threonine kinase ubiquitously expressed in most cell types,appears to mediate or potentiate apoptosis due to many stimuli thatactivate the mitochondrial cell death pathway. Loberg, R D, et al., J.Biol. Chem. 277 (44): 41667-673 (2002). It has been demonstrated toinduce caspase 3 activation and to activate the proapoptotic tumorsuppressor gene p53. It also has been suggested that GSK-3 promotesactivation and translocation of the proapoptotic Bcl-2 family member,Bax, which, upon aggregation and mitochondrial localization, inducescytochrome c release. Akt is a critical regulator of GSK-3, andphosphorylation and inactivation of GSK-3 may mediate some of theantiapoptotic effects of Akt.

The term “assay marker” or “reporter gene” (or “reporter”) refers to agene that can be detected, or easily identified and measured. Theexpression of the reporter gene may be measured at either the RNA level,or at the protein level. The gene product, which may be detected in anexperimental assay protocol, includes, but is not limited to, markerenzymes, antigens, amino acid sequence markers, cellular phenotypicmarkers, nucleic acid sequence markers, and the like. Researchers mayattach a reporter gene to another gene of interest in cell culture,bacteria, animals, or plants. For example, some reporters are selectablemarkers, or confer characteristics upon on organisms expressing themallowing the organism to be easily identified and assayed. To introducea reporter gene into an organism, researchers may place the reportergene and the gene of interest in the same DNA construct to be insertedinto the cell or organism. For bacteria or eukaryotic cells in culture,this may be in the form of a plasmid. Commonly used reporter genes mayinclude, but are not limited to, fluorescent proteins, luciferase,beta-galactosidase, and selectable markers, such as chloramphenicol andkanomycin.

As used herein, the term “bioavailability” refers to the rate and extentto which the active drug ingredient or therapeutic moiety is absorbedinto the systemic circulation from an administered dosage form ascompared to a standard or control.

The term “biomarkers” (or “biosignatures”) as used herein refers topeptides, proteins, nucleic acids, antibodies, genes, metabolites, orany other substances used as indicators of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention. Theterm “indicator” as used herein refers to any substance, number or ratioderived from a series of observed facts that may reveal relative changesas a function of time; or a signal, sign, mark, note or symptom that isvisible or evidence of the existence or presence thereof. Once aproposed biomarker has been validated, it may be used to diagnosedisease risk, presence of disease in an individual, or to tailortreatments for the disease in an individual (choices of drug treatmentor administration regimes). In evaluating potential drug therapies, abiomarker may be used as a surrogate for a natural endpoint, such assurvival or irreversible morbidity. If a treatment alters the biomarker,and that alteration has a direct connection to improved health, thebiomarker may serve as a surrogate endpoint for evaluating clinicalbenefit. Clinical endpoints are variables that can be used to measurehow patients feel, function or survive. Surrogate endpoints arebiomarkers that are intended to substitute for a clinical endpoint;these biomarkers are demonstrated to predict a clinical endpoint with aconfidence level acceptable to regulators and the clinical community.

The term “bound” or any of its grammatical forms as used herein refersto the capacity to hold onto, attract, interact with or combine with.

The terms “cancer” or “malignancy” as used herein refer to diseases inwhich abnormal cells divide without control and can invade nearbytissues. Cancer cells also can spread to other parts of the body throughthe blood and lymph systems. There are several main types of cancer.Carcinoma is a cancer that begins in the skin or in tissues that line orcover internal organs. Sarcoma is a cancer that begins in bone,cartilage, fat, muscle, blood vessels, or other connective or supportivetissue. Leukemia is a cancer that starts in blood-forming tissue such asthe bone marrow, and causes large numbers of abnormal blood cells to beproduced and enter the blood. Lymphoma and multiple myeloma are cancersthat begin in cells of the immune system. Central nervous system cancersare cancers that begin in the tissues of the brain and spinal cord.

The term “carrier” as used herein describes a material that does notcause significant irritation to an organism and does not abrogate thebiological activity and properties of the active compound of thecomposition of the described invention. Carriers must be of sufficientlyhigh purity and of sufficiently low toxicity to render them suitable foradministration to the mammal being treated. The carrier can be inert, orit can possess pharmaceutical benefits, cosmetic benefits or both. Theterms “excipient”, “carrier”, or “vehicle” are used interchangeably torefer to carrier materials suitable for formulation and administrationof pharmaceutically acceptable compositions described herein. Carriersand vehicles useful herein include any such materials know in the artwhich are nontoxic and do not interact with other components.

The term “cell” is used herein to refer to the structural and functionalunit of living organisms and is the smallest unit of an organismclassified as living.

The term “cell line” as used herein refers to a population ofimmortalized cells, which have undergone transformation and can bepassed indefinitely in culture.

The term “chemoresistance” as used herein refers to the development of acell phenotype resistant to a variety of structurally and functionallydistinct agents. Tumors can be intrinsically resistant prior tochemotherapy, or resistance may be acquired during treatment by tumorsthat are initially sensitive to chemotherapy. Drug resistance is amultifactorial phenomenon involving multiple interrelated or independentmechanisms. A heterogeneous expression of involved mechanisms maycharacterize tumors of the same type or cells of the same tumor and mayat least in part reflect tumor progression. Exemplary mechanisms thatcan contribute to cellular resistance include: increased expression ofdefense factors involved in reducing intracellular drug concentration;alterations in drug-target interaction; changes in cellular response, inparticular increased cell ability to repair DNA damage or toleratestress conditions, and defects in apoptotic pathways.

The term “chemosensitive”, “chemosensitivity” or “chemosensitive tumor”as used herein refers to a tumor that is responsive to a chemotherapy ora chemotherapeutic agent. Characteristics of a chemosensitive tumorinclude, but are not limit to, reduced proliferation of the populationof tumor cells, reduced tumor size, reduced tumor burden, tumor celldeath, and slowed/inhibited progression of the population of tumorcells.

The term “chemotherapeutic agent” as used herein refers to chemicalsuseful in the treatment or control of a disease, e.g., cancer

The term “chemotherapy” as used herein refers to a course of treatmentwith one or more chemotherapeutic agents. In the context of cancer, thegoal of chemotherapy is, e.g., to kill cancer cells, reduceproliferation of cancer cells, reduce growth of a tumor containingcancer cells, reduce invasiveness of cancer cells, increase apoptosis ofcancer cells.

The term “chemotherapy regimen” (“combination chemotherapy”) meanschemotherapy with more than one drug in order to benefit from thedissimilar toxicities of the more than one drug. A principle ofcombination cancer therapy is that different drugs work throughdifferent cytotoxic mechanisms; since they have different dose-limitingadverse effects, they can be given together at full doses.

The term “compatible” as used herein means that the components of acomposition are capable of being combined with each other in a mannersuch that there is no interaction that would substantially reduce theefficacy of the composition under ordinary use conditions.

The term “condition”, as used herein, refers to a variety of healthstates and is meant to include disorders or diseases caused by anyunderlying mechanism or injury.

The term “contact” and its various grammatical forms as used hereinrefers to a state or condition of touching or of immediate or localproximity. Contacting a composition to a target destination, such as,but not limited to, an organ, a tissue, a cell, or a tumor, may occur byany means of administration known to the skilled artisan.

The term “derivative” as used herein means a compound that may beproduced from another compound of similar structure in one or moresteps. A “derivative” or “derivatives” of a peptide or a compoundretains at least a degree of the desired function of the peptide orcompound. Accordingly, an alternate term for “derivative” may be“functional derivative.” Derivatives can include chemical modificationsof the peptide, such as akylation, acylation, carbamylation, iodinationor any modification that derivatives the peptide. Such derivatizedmolecules include, for example, those molecules in which free aminogroups have been derivatized to form amine hydrochlorides, p-toluenesulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,chloroacetyl groups or formal groups. Free carboxyl groups can bederivatized to form salts, esters, amides, or hydrazides. Free hydroxylgroups can be derivatized to form O-acyl or O-alkyl derivatives. Theimidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. Also included as derivatives or analogues arethose peptides that contain one or more naturally occurring amino acidderivative of the twenty standard amino acids, for example,4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, homoserine,ornithine or carboxyglutamiate, and can include amino acids that are notlinked by peptide bonds. Such peptide derivatives can be incorporatedduring synthesis of a peptide, or a peptide can be modified by wellknownchemical modification methods (see, e.g., Glazer et al., ChemicalModification of Proteins, Selected Methods and Analytical Procedures,Elsevier Biomedical Press, New York (1975)).

The term “detectable marker” encompasses both selectable markers andassay markers. The term “selectable markers” refers to a variety of geneproducts to which cells transformed with an expression construct can beselected or screened, including drug-resistance markers, antigenicmarkers useful in fluorescence-activated cell sorting, adherence markerssuch as receptors for adherence ligands allowing selective adherence,and the like. When a nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed.

The term “detectable response” refers to any signal or response that maybe detected in an assay, which may be performed with or without adetection reagent. Detectable responses include, but are not limited to,radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared,visible) emission, absorption, polarization, fluorescence,phosphorescence, transmission, reflection or resonance transfer.Detectable responses also include chromatographic mobility, turbidity,electrophoretic mobility, mass spectrum, ultraviolet spectrum, infraredspectrum, nuclear magnetic resonance spectrum and x-ray diffraction.Alternatively, a detectable response may be the result of an assay tomeasure one or more properties of a biologic material, such as meltingpoint, density, conductivity, surface acoustic waves, catalytic activityor elemental composition.

The term “disease” or “disorder”, as used herein, refers to animpairment of health or a condition of abnormal functioning.

The term “dose” as used herein refers to the quantity of medicineprescribed to be taken at one time.

The term “drug” as used herein refers to a therapeutic agent or anysubstance used in the prevention, diagnosis, alleviation, treatment, orcure of disease.

The terms “Emopamil Binding Protein” (EBP), “Human Sterol Isomerase”(HIS) and “delta8-delta7 sterol isomerase” are used interchangeably torefer to an integral membrane protein of the endoplasmic reticulum thatcatalyzes the conversion of delta(8)-sterols into delta(7)-sterols.

The term “effective amount” or “amount effective” refers to the amountnecessary or sufficient to realize a desired biologic effect.

The term “effective dose” as used herein refers to the quantity ofmedicine prescribed to be taken at one time necessary or sufficient torealize a desired biologic effect.

As used herein, the term “enzymatic activity” refers to the amount ofsubstrate consumed (or product formed) in a given time under givenconditions. Enzymatic activity also may be referred to as “turnovernumber.”

As used herein, the terms “formulation” and “composition” are usedinterchangeably to refer to a product of the described invention thatcomprises all active and inert ingredients.

The term “functional equivalent” or “functionally equivalent” are usedinterchangeably herein to refer to substances, molecules,polynucleotides, proteins, peptides, or polypeptides having similar oridentical biological activity to a reference substance, molecule,polynucleotide, protein, peptide, or polypeptide. Any small moleculeanti-cancer compound that retains the biological activity of compound4C12, e.g. modulating a cancer cell sensitive to cholesterolbiosynthesis inhibition, may be used as such a functional equivalent.

The term “growth” as used herein refers to a process of becoming larger,longer or more numerous, or an increase in size, number, or volume.

The term “half maximal inhibitory concentration” (“IC50”) is a measureof the effectiveness of a compound in inhibiting a biological orbiochemical function.

The terms “inhibiting”, “inhibit” or “inhibition” are used herein torefer to reducing the amount or rate of a process, to stopping theprocess entirely, or to decreasing, limiting, or blocking the action orfunction thereof. Inhibition may include a reduction or decrease of theamount, rate, action function, or process of a substance by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%.

The term “inhibitor” as used herein refers to a molecule that binds toan enzyme thereby decreasing enzyme activity. Enzyme inhibitors aremolecules that bind to enzymes thereby decreasing enzyme activity. Thebinding of an inhibitor may stop substrate from entering the active siteof the enzyme and/or hinder the enzyme from catalyzing its reaction.Inhibitor binding is either reversible or irreversible. Irreversibleinhibitors usually react with the enzyme and change it chemically, forexample, by modifying key amino acid residues needed for enzymaticactivity. In contrast, reversible inhibitors bind non-covalently andproduce different types of inhibition depending on whether theseinhibitors bind the enzyme, the enzyme-substrate complex, or both.Enzyme inhibitors often are evaluated by their specificity and potency.

The term “injury,” as used herein, refers to damage or harm to astructure or function of the body caused by an outside agent or force,which may be physical or chemical.

The term “interfere” or “to interfere with” as used herein refers to thehampering, impeding, dampening, hindering, obstructing, blocking,reducing or preventing of an action or occurrence. By way of example, areceptor antagonist interferes with (e.g., blocks or dampens) anagonist-mediated response rather than provoking a biological responseitself.

The term “invasion” or “invasiveness” as used herein refers to a processin malignant cells that includes penetration of and movement throughsurrounding tissues.

The term “Kaplan Meier plot” or “Kaplan Meier survival curve” as usedherein refers to the plot of probability of clinical study subjectssurviving in a given length of time while considering time in many smallintervals. The Kaplan Meier plot assumes that: (i) at any time subjectswho are censored (i.e., lost) have the same survival prospects assubjects who continue to be followed; (ii) the survival probabilitiesare the same for subjects recruited early and late in the study; and(iii) the event (e.g., death) happens at the time specified.Probabilities of occurrence of events are computed at a certain point oftime with successive probabilities multiplied by any earlier computedprobabilities to get a final estimate. The survival probability at anyparticular time is calculated as the number of subjects survivingdivided by the number of subjects at risk. Subjects who have died,dropped out, or have been censored from the study are not counted as atrisk.

The term “ligand” as used herein refers to a molecule that can bindselectively to a molecule, such that the binding interaction between theligand and its binding partner is detectable over nonspecificinteractions by a quantifiable assay. Derivatives, analogues and mimeticcompounds are intended to be included within the definition of thisterm.

The terms “marker” and “cell surface marker” are used interchangeablyherein to refer to a receptor, a combination of receptors, or anantigenic determinant or epitope found on the surface of a cell thatallows a cell type to be distinguishable from other kinds of cells.Specialized protein receptors (markers) that have the capability ofselectively binding or adhering to other signaling molecules coat thesurface of every cell in the body. Cells use these receptors and themolecules that bind to them as a way of communicating with other cellsand to carry out their proper function in the body. Cell sortingtechniques are based on cellular biomarkers where a cell surfacemarker(s) may be used for either positive selection or negativeselection, i.e., for inclusion or exclusion, from a cell population.

The term “maximum tolerated dose” (MTD) as used herein refers to thehighest dose of a drug that does not produce unacceptable toxicity.

The term “median survival” as used herein refers to the time after which50% of individuals with a particular condition are still living and 50%have died. For example, a median survival of 6 months indicates thatafter 6 months, 50% of individuals with, e.g., colon cancer would bealive, and 50% would have passed away. Median survival is often used todescribe the prognosis (i.e., chance of survival) of a condition whenthe average survival rate is relatively short, such as for colon cancer.Median survival is also used in clinical studies when a drug ortreatment is being evaluated to determine whether or not the drug ortreatment will extend life.

The term “metastasis” as used herein refers to the transference oforganisms or of malignant or cancerous cells, producing diseasemanifestations, from one part of the body to other parts.

The term “migration” as used herein refers to a movement of a populationof cells from one place to another.

The term “mitotic index” as used herein refers to the ratio of thenumber of cells undergoing mitosis (cell division) to the number ofcells not undergoing mitosis in a population of cells.

The term “modify” as used herein means to change, vary, adjust, temper,alter, affect or regulate to a certain measure or proportion in one ormore particulars.

The term “modifying agent” as used herein refers to a substance,composition, therapeutic component, active constituent, therapeuticagent, drug, metabolite, active agent, protein, non-therapeuticcomponent, non-active constituent, non-therapeutic agent, or non-activeagent that reduces, lessens in degree or extent, or moderates the form,symptoms, signs, qualities, character or properties of a condition,state, disorder, disease, symptom or syndrome.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “neoplasm” as used herein refers to an abnormal proliferationof genetically altered cells. A malignant neoplasm (or malignant tumor)is synonymous with cancer. A benign neoplasm (or benign tumor) is atumor (solid neoplasm) that stops growing by itself, does not invadeother tissues and does not form metastases.

The term “neurosphere” as used herein refers to three-dimensionalaggregates of cells in suspension when cultured in serum-free conditionssupplemented with epidermal growth factor (EGF) and basic fibroblastgrowth factor (bFGF) that, can be dissociated to form numerous secondaryspheres or, when grown in differentiating medium on an appropriatesubstrate, induced to differentiate, generating neurons, astrocytes andoligodendrocytes, the three major cell types of the CNS.

The term “normal healthy control subject” as used herein refers to asubject having no symptoms or other clinical evidence of a disease.

The term “outcome” as used herein refers to a specific result or effectthat can be measured. Nonlimiting examples of outcomes include decreasedpain, reduced tumor size, and survival (e.g., progression-free survivalor overall survival).

The term “overall survival” (OS) as used herein refers to the length oftime from either the date of diagnosis or the start of treatment for adisease, such as cancer, that patients diagnosed with the disease arestill alive.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle); intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord or under the arachnoid membrane of thebrain), or infusion techniques. A parenterally administered compositionis delivered using a needle, e.g., a surgical needle. The term “surgicalneedle” as used herein, refers to any needle adapted for delivery offluid (i.e., capable of flow) compositions into a selected anatomicalstructure. Injectable preparations, such as sterile injectable aqueousor oleaginous suspensions, may be formulated according to the known artusing exemplary dispersing or wetting agents and suspending agents.

The terms “pharmaceutical formulation” or “pharmaceutical composition”as used herein refer to a formulation or composition that is employed toprevent, reduce in intensity, cure or otherwise treat a target conditionor disease.

The term “pharmaceutically acceptable carrier” as used herein refers toany substantially non-toxic carrier conventionally useable foradministration of pharmaceuticals in which the small moleculeanti-cancer compound of the described invention will remain stable andbioavailable. The pharmaceutically acceptable carrier must be ofsufficiently high purity and of sufficiently low toxicity to render itsuitable for administration to the mammal being treated. It furthershould maintain the stability and bioavailability of an active agent.The pharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition.

The term “pharmaceutically acceptable salt” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. When used inmedicine the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof. Such salts include,but are not limited to, those prepared from the following acids:hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene sulphonic, tartaric, citric, methanesulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, andbenzene sulphonic. Also, such salts may be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts of thecarboxylic acid group. By “pharmaceutically acceptable salt” is meantthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well-known in the art. For example, P. H. Stahl, etal. describe pharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002). The salts may be prepared in situ during thefinal isolation and purification of the compounds described within thedescribed invention or separately by reacting a free base function witha suitable organic acid. Representative acid addition salts include, butare not limited to, acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptanoate,hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides likebenzyl and phenethyl bromides and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsalso may be obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound such as an aminewith a suitable acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

The terms “primary tumor” or “primary cancer” are used interchangeablyto refer to the original, or first, tumor in the body. Cancer cells froma primary cancer may spread to other parts of the body and form new, orsecondary tumors. This is called metastasis. The secondary tumors arethe same type of cancer as the primary cancer.

The term “progression” as used herein refers to the course of a diseaseas it becomes worse or spreads in the body.

The term “progression-free survival” (PFS) as used herein refers to thelength of time during and after the treatment of a disease that apatient lives with the disease but it does not get worse.

The term “proliferation” as used herein refers to expansion of apopulation of cells by the continuous division of single cells intoidentical daughter cells, leading to a multiplying or increasing in thenumber of cells.

The term “recurrence” as used herein refers to a disease (e.g., cancer)that has come back, usually after a period of time during which thedisease could not be detected.

The term “reduce” or “reducing” as used herein refers to limitoccurrence of a disorder in individuals at risk of developing thedisorder.

The terms “refractory” or “resistant” are used interchangeably hereinrefers to a disease or condition that does not respond to treatment. Thedisease may be resistant at the beginning of treatment or it may becomeresistant during treatment.

The term “remission” as used herein refers to a decrease in ordisappearance of signs and symptoms of a disease. In partial remission,some, but not all, signs and symptoms have disappeared. In completeremission, all signs and symptoms have disappeared although the diseasemay still be in the body.

The term Response Evaluation Criteria in Solid Tumors (or “RECIST”) asused herein refers to a standard way to measure how well a cancerpatient responds to treatment. It is based on whether tumors shrink,stay the same, or get bigger. To use RECIST, there must be at least onetumor that can be measured on x-rays, CT scans, or MRI scans. The typesof response a patient can have are a complete response (CR), a partialresponse (PR), progressive disease (PD), and stable disease (SD).

The term “sign” as used herein refers to something found during aphysical exam or from a laboratory test that shows that a person mayhave a condition or disease.

The terms “subject” or “individual” or “patient” are usedinterchangeably to refer to a member of an animal species of mammalianorigin, including but not limited to, a mouse, a rat, a cat, a goat,sheep, horse, hamster, ferret, platypus, pig, a dog, a guinea pig, arabbit and a primate, such as, for example, a monkey, ape, or human.

The term “subject in need of such treatment” as used herein refers to apatient who suffers from a disease, disorder, condition, or pathologicalprocess, e.g., a solid tumor, a brain cancer, or a glioma. According tosome embodiments, the term “subject in need of such treatment” also isused to refer to a patient whose cancer comprises a population of cancercells sensitive to cholesterol biosynthesis inhibition; (i) who will beadministered a therapeutic amount of a small molecule anti-cancercompound of the described invention (ii) is receiving a therapeuticamount of a small molecule anti-cancer compound of the describedinvention; or (iii) has received a therapeutic amount of a smallmolecule anti-cancer compound of the described invention unless thecontext and usage of the phrase indicates otherwise.

The terms “substantial inhibition”, “substantially inhibited” and thelike as used herein refer to inhibition of at least 50%, inhibition ofat least 55%, inhibition of at least 60%, inhibition of at least 65%,inhibition of at least 70%, inhibition of at least 75%, inhibition of atleast 80%, inhibition of at least 85%, inhibition of at least 90%,inhibition of at least 95%, or inhibition of at least 99%.

The term “survival rate” as used herein refers to the percent ofindividuals who survive a disease (e.g., cancer) for a specified amountof time. For example, if the 5-year survival rate for a particularcancer is 34%, this means that 34 out of 100 individuals initiallydiagnosed with that cancer would be alive after 5 years.

The term “symptom” as used herein refers to a sign or a disease orcondition. The terms “symptom management”, “palliative care,” and“supportive care” are used interchangeably herein to refer to care givento improve the quality of life (QOL) of patients who have a serious orlife-threatening disease.

The term “therapeutic agent” as used herein refers to a drug, molecule,nucleic acid, protein, metabolite, composition or other substance thatprovides a therapeutic effect. The term “active” as used herein refersto the ingredient, component or constituent of the compositions of thedescribed invention responsible for the intended therapeutic effect. Theterms “therapeutic agent” and “active agent” are used interchangeablyherein.

The terms “therapeutically effective amount”, an “amount effective”, or“pharmaceutically effective amount” of one or more of the active agentsand used interchangeably to refer to an amount that is sufficient toprovide the intended benefit of treatment. Dosage levels are based on avariety of factors, including the type of injury, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular active agent employed. Thus,the dosage regimen may vary widely, but can be determined routinely by aphysician using standard methods. Additionally, the terms“therapeutically effective amounts” and “pharmaceutically effectiveamounts” include prophylactic or preventative amounts.

The term “therapeutic component” as used herein refers to atherapeutically effective dosage (i.e., dose and frequency ofadministration) that eliminates, reduces, or prevents the progression ofa particular disease manifestation in a percentage of a population. Anexample of a commonly used therapeutic component is the ED50, whichdescribes the dose in a particular dosage that is therapeuticallyeffective for a particular disease manifestation in 50% of a population.

The term “therapeutic effect” as used herein refers to a consequence oftreatment, the results of which are judged to be desirable andbeneficial. A therapeutic effect may include, directly or indirectly,the arrest, reduction, or elimination of a disease manifestation. Atherapeutic effect may also include, directly or indirectly, the arrestreduction or elimination of the progression of a disease manifestation.

The term “sterol” as used herein refers to a steroid alcohol, whichcontains a common steroid nucleus (a fused, reduced 17-carbon-atom ringsystem, cyclopentanoperhydrophenantrene) and a hydroxyl group.

As used herein the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical symptoms of a condition, orsubstantially preventing the appearance of clinical symptoms of acondition. Treating further refers to accomplishing one or more of thefollowing: (a) reducing the severity of the disorder; (b) limitingdevelopment of symptoms characteristic of the disorder(s) being treated;(c) limiting worsening of symptoms characteristic of the disorder(s)being treated; (d) limiting recurrence of the disorder(s) in patientsthat have previously had the disorder(s); and (e) limiting recurrence ofsymptoms in patients that were previously asymptomatic for thedisorder(s).

The term “tumor” as used herein refers to a diseases involving abnormalcell growth in numbers (proliferation) or in size with the potential toinvade or spread to other parts of the body (metastasis).

The term “tumor burden” or “tumor load” are used interchangeably hereinrefers to the number of cancer cells, the size of a tumor, or the amountof cancer in the body.

Compounds

A small molecule anti-cancer compound of Formula I:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

Y¹ is selected from the group consisting of C═O, CH₂, and SO₂;

n=1, 2, or 3;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et,isopropyl, cyclopropyl, and C₂-C₆ alkynyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to one embodiment, the described invention provides a smallmolecule anti-cancer compound of Formula I wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

Y¹ is selected from the group consisting of C═O, CH₂, and SO₂;

n=1 or 2;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆hydroxyalkyl, C₁-C₆ alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁵ and R⁶ are independently selected from the group consisting ofcycloalkyl, aryl, heteroaryl, heterocyclyl, benzyl, fusedheteroarylaryl, fused arylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

Y¹ is selected from the group consisting of C═O, CH₂, and SO₂;

n=1 or 2;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁵ and R⁶ are independently selected from the group consisting ofcycloalkyl, aryl, heteroaryl, heterocyclyl, benzyl, fusedheteroarylaryl, fused arylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

X²=L²-R⁶;

Y¹ is selected from the group consisting of C═O, CH₂, and SO₂;

n=1 or 2;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁵ and R⁶ are independently selected from the group consisting ofcycloalkyl, aryl, heteroaryl, heterocyclyl, benzyl, fusedheteroarylaryl, fused arylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-a:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

n=1, 2, or 3;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl,cyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-a wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

n=1 or 2;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises —(CRwhere m=2, 3, 4, or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-a wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L¹-R⁵;

X² is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,SO₂Me, and L²-R⁶;

n=1 or 2;

L¹ is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises —(CRwhere m=2, 3, 4, or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁵ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-a wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

X²=L²-R⁶;

n=1 or 2;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4, or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, propargyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-b:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-b wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1 or 2;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₆ alkyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides smallmolecule anti-cancer compound of Formula I-c:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y² is selected from the group consisting of CR′R″, NR, O, and S. In thecontext of this paragraph, R, R′ and R″ are independently selected fromthe group consisting of H, F, Me, Et, i-Pr, and cyclopropyl;

k=0, 1, 2, or 3;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-c wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y² is selected from the group consisting of CH₂, NR, O, and S. In thecontext of this paragraph, R is selected from the group consisting of Hand Me;

k=1 or 2;

n=1 or 2;

L² is selected from the group consisting of NH, O, S, CHOH, C═O, and—S(CH₂)—;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-c wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y² is selected from the group consisting of CH₂, NR, O, and S. In thecontext of this paragraph, R is selected from the group consisting of Hand Me;

k=1 or 2;

n=1 or 2;

L² is selected from the group consisting of NH, O, and S;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁶ is selected from the group consisting of aryl, and heteroaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides smallmolecule anti-cancer compound of Formula I-d:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y³ is selected from the group consisting of CH₂, NR, O, and S. In thecontext of this paragraph, R is selected from the group consisting of H,Me, CD₃, CF₃, Et, isopropyl, and cyclopropyl.

n=1 or 2;

L² is selected from the group consisting of S, O, NH;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, andC₂-C₆ alkynyl;

R⁶ is selected from the group consisting of aryl, heteroaryl, benzyl,fused heteroarylaryl, fused arylheteroaryl, and fused arylaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-d wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y³ is selected from the group consisting of CH₂, NH, NMe, and O;

n=1 or 2;

L² is selected from the group consisting of S, O, and NH;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁶ is selected from the group consisting of aryl, and heteroaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-e:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y⁴ is selected from the group consisting of CR′R″, NR, O, and S. In thecontext of this paragraph, R, R′, and R″ are independently selected fromthe group consisting of H, F, Me, Et, isopropyl and cyclopropyl;

j=0, 1, 2, or 3;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-e wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

Y⁴═CH₂;

j=1;

n=1 or 2;

L² is selected from the group consisting of S, O, NH, CHOH, C═O, and—S(CH₂)—;

R² is selected from the group consisting of H, C₁-C₃ alkyl, propargyl,C₁-C₃ hydroxyalkyl, and acyloxyalkyl;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R⁶ is selected from the group consisting of cycloalkyl, aryl,heteroaryl, heterocyclyl, benzyl, fused heteroarylaryl, fusedarylheteroaryl, and fused arylaryl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-f:

Wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1, 2, or 3;

L² is selected from the group consisting of S, O, NH, CHOH, C═O,—O(CH₂)—, —S(CH₂)—, —(CH₂)O—, and —(CH₂)S—;

R¹ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of athree to six subunit chain comprising subunits independently selectedfrom the group consisting of (CR⁷R⁸), NR⁹, O, and S;

R¹ and R³ may optionally form a ring, such that R¹-R³ comprises—(CR¹⁰R¹¹)_(m)—, where m=2, 3, 4 or 5;

R² is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, cycloalkyl, C₁-C₆ hydroxyalkyl, C₁-C₆alkoxyalkyl, and acyloxyalkyl;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a oneto four subunit chain comprising subunits independently selected fromthe group consisting of —(CR¹²R¹³)—. Additionally, R² may simultaneouslyform a ring with R¹ as described above;

R³ is selected from the group consisting of H, D, F, Me, and Et;

R⁴ is selected from the group consisting of H, Me, CD₃, CF₃, Et, i-Pr,cyclopropyl, and C₂-C₆ alkynyl;

R⁷ and R⁸ are independently selected from the group consisting of H, D,F, Me, Et, OR, and NR₂. In the context of this paragraph, R is selectedfrom the group consisting of H, Me, and Et;

R⁹ is selected from the group consisting of H, Me, Et, isopropyl, andcyclopropyl;

R¹⁰ and R¹¹ are independently selected from the group consisting of H,D, F, Me, Et, OR, and NR₂. In the context of this paragraph, R isselected from the group consisting of H, Me, and Et;

R¹² and R¹³ are independently selected from the group consisting of H,D, F, Me, and Et;

R¹⁴ and R¹⁵ can be attached at any available position on the aromaticring and are selected from the group consisting of H, D, F, Cl, Br, CF₃,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, cycloalkyl, OR, NR₂, NO₂, N₃,CN, CO₂R, CO₂NR₂, SR, alkylacyl and arylacyl. In the context of thisparagraph, R is independently selected from the group consisting of H,Me, Et, isopropyl, cyclopropyl, propargyl, and acyl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

According to another embodiment, the described invention provides asmall molecule anti-cancer compound of Formula I-f wherein:

X¹ is selected from the group consisting of H, F, Cl, CN, NH₂, NO₂, N₃,and SO₂Me;

n=1 or 2;

L² is selected from the group consisting of S, O, NH, CHOH, C═O, and—S(CH₂)—;

R¹ and R² are independently selected from the group consisting of H,C₁-C₃ alkyl, propargyl, C₁-C₃ hydroxyalkyl, and acyloxyalkyl;

R¹ and R² may optionally form a ring, such that R¹-R² consists of a fouror five subunit chain comprising subunits independently selected fromthe group consisting of CH₂, NH, NMe, and O;

R¹ and R³ may optionally form a ring, such that R¹-R³═—(CH₂)₃—;

R² and R⁴ may optionally form a ring, such that R²-R⁴ consists of a twosubunit chain comprising subunits independently selected from the groupconsisting of CH₂ and CH-alkyl. Additionally, R² may simultaneously forma ring with R¹ as described above;

R³═H;

R⁴ is selected from the group consisting of H, Me, and propargyl;

R¹⁴ and R¹⁵ can be attached at any available position on the aromaticring and are selected from the group consisting of H, F, Cl, Br, CF₃,C₁-C₃ alkyl, propargyl, OR, NR₂, NO₂, CN, CO₂R, CO₂NR₂, and SR. In thecontext of this paragraph, R is independently selected from the groupconsisting of H, Me, Et, and propargyl;

Such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present;

or a pharmaceutically acceptable salt, prodrug, active metabolite, orsolvate thereof.

Chemical Substituents

The term “Aliphatic” as used herein, denotes a straight- orbranched-chain arrangement of constituent carbon atoms, including, butnot limited to paraffins (alkanes), which are saturated, olefins(alkenes or alkadienes), which are unsaturated, and acetylenes(alkynes), which contain a triple bond. In complex structures, thechains may be branched or cross-linked.

The term “lower” as used herein refers to a group having between one andsix carbons.

As used herein, the term “alkyl” refers to a straight or branched chainhydrocarbon having from 1 to 25 carbon atoms, or of the numbers ofcarbon atoms specified (e.g. C₁₋₆ alkyl) or any numbers within thisrange. It is implicitly implied within the context of this applicationthat such alkyl groups can be optionally substituted with substituentssuch as, but not limited to, halogen, perfluoroalkyl, lower alkyl, loweralkenyl, lower alkynyl, lower cycloalkyl, lower alkoxy, lowercycloalkoxy, lower alkylsulfanyl, oxo, hydroxyl. Examples of “alkyl” asused herein include, but are not limited to, methyl, trifluoromethyl,ethyl, propyl, n-butyl, t-butyl, n-pentyl, isobutyl, and isopropyl,methoxymethy, methoxyethyl, isopropoxybutyl, propynyloxyethyl, and thelike.

The term “Alkenyl,” as used herein, denotes a monovalent, straight(unbranched) or branched hydrocarbon chain having one or more doublebonds therein where the double bond can be unconjugated or conjugated toanother unsaturated group (e.g., a polyunsaturated alkenyl) and can beunsubstituted or substituted, with multiple degrees of substitutionbeing allowed. It may be optionally substituted with substituents suchas, but not limited to, halogen, perfluoroalkyl, lower alkyl, loweralkenyl, lower alkynyl, lower cycloalkyl, lower alkoxy, lowercycloalkoxy, lower alkylsulfanyl, oxo, hydroxyl. For example, andwithout limitation, the alkenyl can be vinyl, allyl, butenyl, pentenyl,hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl,2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl, 6-methoxyhexenyl,2-trifluoromethyl-3-butenyl, and the like.

As used herein, the term “alkynyl” refers to a hydrocarbon radicalhaving at least one carbon-carbon triple bond, optionally substitutedwith substituents such as, without limitation, halogen, perfluoroalkyl,lower alkyl, lower alkenyl, lower alkynyl, lower cycloalkyl, loweralkoxy, lower cycloalkoxy, lower alkylsulfanyl, oxo, hydroxyl.

The term “aryl” as used herein refers to a benzene ring or to anoptionally substituted benzene ring system fused to one or moreoptionally substituted benzene rings, with multiple degrees ofsubstitution being allowed. Substituents include, but are not limitedto, cyano, halogen, perfluoroalkyl, aryl, heteroaryl, heterocyclyl,cycloalkyl, lower alkyl, lower alkoxy, lower alkylsulfanyl, oxo,hydroxy, amino optionally substituted by alkyl or aryl or heteroaryl orheterocyclyl or cycloalkyl, aminocarbonyl (—NRC(O)R) optionallysubstituted by alkyl or aryl or heteroaryl or heterocyclyl orcycloalkyl, carboxy, acyl, acyloxy, alkoxycarbonyl, aryloxy,heteroaryloxy, heterocyclyloxy, aroyloxy, heteroaroyloxy,heterocycloyloxy, carbamoyl optionally substituted by alkyl orcycloalkyl or aryl or heteroaryl or heterocyclyl, aminosulfonyloptionally substituted by alkyl or cycloalkyl or aryl or heteroaryl orheterocyclyl. Examples of aryl include, but are not limited to, phenyl,2-napthyl, 1-naphthyl, 1-anthracenyl, and the like.

It should be understood that wherever the terms “alkyl” or “aryl” oreither of their prefix roots appear in a name of a substituent, they areto be interpreted as including those limitations given above for alkyland aryl. Designated numbers of carbon atoms (e.g. C₁₋₁₀) shall referindependently to the number of carbon atoms in an alkyl, alkenyl oralkynyl or cyclic alkyl moiety or to the alkyl portion of a largersubstituent in which the term “alkyl” appears as its prefix root.

As used herein, “cycloalkyl” (used interchangeably with “aliphaticcyclic” herein) refers to a non-aromatic monovalent, monocyclic orpolycyclic ring structure having a total of from 3 to 10 carbon ringatoms (but no heteroatoms) optionally possessing one or more degrees ofunsaturation, optionally substituted with substituents such as, withoutlimitation, halogen, perfluoroalkyl, cycloalkyl, lower alkyl, loweralkoxy, lower alkylsulfanyl, oxo, hydroxyl. “Cycloalkyl” includes by wayof example cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohehexenyl, adamantanyl, norbornyl, nobornenyl,cycloheptyl, or cyclooctyl, and the like.

The terms “heterocycle” and “heterocyclic” as used herein are usedinterchangeably to refer to a three to twelve-membered heterocyclic ringoptionally possessing one or more degrees of unsaturation, containingone or more heteroatomic substitutions selected from —S—, —SO—, —SO₂—,—O—, or —N—, optionally substituted with substitutents, including, butnot limited to, nitro, cyano, halogen, perfluoroalkyl, aryl, heteroaryl,heterocyclyl, cycloalkyl, lower alkyl, lower alkoxy, loweralkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo, hydroxy,mercapto, amino optionally substituted by alkyl or aryl or heteroaryl orheterocyclyl or cycloalkyl, aminocarbonyl (—NRC(O)R) optionallysubstituted by alkyl or aryl or heteroaryl or heterocyclyl orcycloalkyl, carboxy, acyl, acyloxy, alkoxycarbonyl, aryloxy,heteroaryloxy, heterocyclyloxy, aroyloxy, heteroaroyloxy,heterocycloyloxy, carbamoyl optionally substituted by alkyl orcycloalkyl or aryl or heteroaryl or heterocyclyl, aminosulfonyloptionally substituted by alkyl or cycloalkyl or aryl or heteroaryl orheterocyclyl, silyloxy optionally substituted by alkyl or aryl, silyloptionally substituted by alkoxy or alkyl or aryl, multiple degrees ofsubstitution being allowed. Such a ring optionally may be fused to oneor more of another “heterocyclic” ring(s). Examples of “heterocyclic”include, but are not limited to, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine,pyrimidine, purine, quinoline, isoquinoline, carbazole, tetrahydrofuran,1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine,piperazine and the like.

Examples of heterocycles include, but are not limited to, pyridyl,thiazolyl, tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl,pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl,tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl,quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, 4aH-carbazolyl, carbazolyl, beta-carbolinyl,phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl,imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl,indolinyl, isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, andisatinoyl.

As used herein, the term “heteroaryl” refers to a five- toseven-membered aromatic ring, or to a polycyclic heterocyclic aromaticring, containing one or more nitrogen, oxygen, or sulfur heteroatoms,where N-oxides and sulfur monoxides and sulfur dioxides are permissibleheteroaromatic substitutions, optionally substituted with substituentsincluding, but not limited to, nitro, cyano, halogen, perfluoroalkyl,aryl, heteroaryl, heterocyclyl, cycloalkyl, lower alkyl, lower alkoxy,lower alkylsulfanyl, lower alkylsulfenyl, lower alkylsulfonyl, oxo,hydroxy, mercapto, amino optionally substituted by alkyl or aryl orheteroaryl or heterocyclyl or cycloalkyl, aminocarbonyl (—NRC(O)R)optionally substituted by alkyl or aryl or heteroaryl or heterocyclyl orcycloalkyl, carboxy, acyl, acyloxy, alkoxycarbonyl, aryloxy,heteroaryloxy, heterocyclyloxy, aroyloxy, heteroaroyloxy,heterocycloyloxy, carbamoyl optionally substituted by alkyl orcycloalkyl or aryl or heteroaryl or heterocyclyl, aminosulfonyloptionally substituted by alkyl or cycloalkyl or aryl or heteroaryl orheterocyclyl, silyloxy optionally substituted by alkyl or aryl, silyloptionally substituted by alkoxy or alkyl or aryl, multiple degrees ofsubstitution being allowed. For polycyclic aromatic ring systems, one ormore of the rings may contain one or more heteroatoms. Examples of“heteroaryl” used herein are furan, thiophene, pyrrole, imidazole,pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole,thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine,quinoline, isoquinoline, quinazoline, benzofuran, benzothiophene,indole, and indazole, and the like.

As used herein, the term “fused cycloalkylaryl” refers to a cycloalkylgroup fused to an aryl group, the two having two atoms in common, andwherein the aryl group is the point of substitution. Examples of “fusedcycloalkylaryl” used herein include, but are not limited to, 5-indanyl,5,6,7,8-tetrahydro-2-naphthyl,

and the like.

As used herein, the term “fused arylcycloalkyl” refers to an aryl groupfused to a cycloalkyl group, the two having two atoms in common, andwherein the cycloalkyl group is the point of substitution. Examples of“fused arylcycloalkyl” used herein include, but are not limited to,1-indanyl, 2-indanyl, 1-(1,2,3,4-tetrahydronaphthyl),

and the like.

As used herein, the term “fused heterocyclylaryl” refers to aheterocyclyl group fused to an aryl group, the two having two atoms incommon, and wherein the aryl group is the point of substitution.Examples of “fused heterocyclylaryl” used herein include, but are notlimited to, 3,4-methylenedioxy-1-phenyl,

and the like.

As used herein, the term “fused arylheterocyclyl” refers to an arylgroup fused to a heterocyclyl group, the two having two atoms in common,and wherein the heterocyclyl group is the point of substitution.Examples of “fused arylheterocyclyl” used herein include, but are notlimited to, 2-(1,3-benzodioxolyl),

and the like.

As used herein, the term “fused cycloalkylheteroaryl” refers to acycloalkyl group fused to a heteroaryl group, the two having two atomsin common, and wherein the heteroaryl group is the point ofsubstitution. Examples of “fused cycloalkylheteroaryl” used hereininclude, but are not limited to, 5-aza-6-indanyl,

and the like.

As used herein, the term “fused heteroarylcycloalkyl” refers to aheteroaryl group fused to a cycloalkyl group, the two having two atomsin common, and wherein the cycloalkyl group is the point ofsubstitution. Examples of “fused heteroarylcycloalkyl” used hereininclude, but are not limited to, 5-aza-1-indanyl,

and the like.

As used herein, the term “fused heterocyclylheteroaryl” refers to aheterocyclyl group fused to a heteroaryl group, the two having two atomsin common, and wherein the heteroaryl group is the point ofsubstitution. Examples of “fused heterocyclylheteroaryl” used hereininclude, but are not limited to, 1,2,3,4-tetrahydro-beta-carbolin-8-yl,

and the like.

As used herein, the term “fused heteroarylheterocyclyl” refers to aheteroaryl group fused to a heterocyclyl group, the two having two atomsin common, and wherein the heterocyclyl group is the point ofsubstitution. Examples of “fused heteroarylheterocyclyl” used hereininclude, but are not limited to, -5-aza-2,3-dihydrobenzofuran-2-yl,

and the like.

As used herein, the term “direct bond”, where part of a structuralvariable specification, refers to the direct joining of the substituentsflanking (preceding and succeeding) the variable taken as a “directbond”.

As used herein, the term “O-linked moiety” means a moiety that is bondedthrough an oxygen atom. Thus, when an R group is an O-linked moiety,that R is bonded through oxygen and it thus can be an ether, an ester(e.g., —O—C(O)-optionally substituted alkyl), a carbonate or a carbamate(e.g., —O—C(O)—NH₂ or —O—C(O)—NH-optionally substituted alkyl).Similarly, the term “S-linked moiety” means a moiety that is bondedthrough a sulfur atom. Thus, when an R group is an S-linked moiety, thatR is bonded through sulfur and it thus can be a thioether (e.g.,—S-optionally substituted alkyl), a thioester (—S—C(O)-optionallysubstituted alkyl) or a disulfide (e.g., —S—S-optionally substitutedalkyl). The term “N-linked moiety” means a moiety that is bonded througha nitrogen atom. Thus, when an R group is an N-linked moiety, the Rgroup is bonded through nitrogen and one or more of these can thus be anN-linked amino acid such as —NH—CH₂—COOH, a carbamate such as—NH—C(O)—O-optionally substituted alkyl, an amine such as —NH-optionallysubstituted alkyl, an amide such as —NH—C(O)-optionally substitutedalkyl or —N₃. The term “C-linked moiety” means a moiety that is bondedthrough a carbon atom. When one or more R group is bonded throughcarbon, one or more of these thus can be -optionally substituted alkylsuch as —C(O)-optionally substituted alkyl hydroxyalkyl, mercaptoalkyl,aminoalkyl or ═CH-optionally substituted alkyl.

The term “alkoxy” as used herein refers to the group R_(a)O—, whereR_(a) is alkyl.

The term “alkenyloxy” as used herein refers to the group R_(a)O—, whereR_(a) is alkenyl.

The term “alkynyloxy” as used herein refers to the group R_(a)O—, whereR_(a) is alkynyl.

The term “alkylsulfanyl” as used herein refers to the group R_(a)S—,where R_(a) is alkyl.

The term “alkenylsulfanyl” as used herein refers to the group R_(a)S—,where R_(a) is alkenyl.

The term “alkynylsulfanyl” as used herein refers to the group R_(a)S—,where R_(a) is alkynyl.

The term “alkylsulfenyl” as used herein refers to the group R_(a)S(O)—,where R_(a) is alkyl.

The term “alkenylsulfenyl” as used herein refers to the groupR_(a)S(O)—, where R_(a) is alkenyl.

The term “alkynylsulfenyl” as used herein refers to the groupR_(a)S(O)—, where R_(a) is alkynyl.

The term “alkylsulfonyl” as used herein refers to the group R_(a)SO₂—,where R_(a) is alkyl.

The term “alkenylsulfonyl” as used herein refers to the group R_(a)SO₂—,where R_(a) is alkenyl.

The term “alkynylsulfonyl” as used herein refers to the group R_(a)SO₂—,where R_(a) is alkynyl.

The term “acyl” as used herein refers to the group R_(a)C(O)—, whereR_(a) is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, or heterocyclyl.

The term “aroyl” as used herein refers to the group R_(a)C(O)—, whereR_(a) is aryl.

The term “heteroaroyl” as used herein refers to the group R_(a)C(O)—,where R_(a) is heteroaryl.

The term “heterocycloyl” as used herein refers to the group R_(a)C(O)—,where R_(a) is heterocyclyl.

The term “alkoxycarbonyl” as used herein refers to the groupR_(a)OC(O)—, where R_(a) is alkyl.

The term “acyloxy” as used herein refers to the group R_(a)C(O)O—, whereR_(a) is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, or heterocyclyl.

The term “aroyloxy” as used herein refers to the group R_(a)C(O)O—,where R_(a) is aryl.

The term “heteroaroyloxy” as used herein refers to the groupR_(a)C(O)O—, where R_(a) is heteroaryl.

The term “heterocycloyloxy” as used herein refers to the groupR_(a)C(O)O—, where R_(a) is heterocyclyl.

The term “substituted” as used herein refers to substitution with thenamed substituent or substituents, multiple degrees of substitutionbeing allowed unless otherwise stated.

The terms “contain” or “containing” can as used herein refers to in-linesubstitutions at any position along the above defined alkyl, alkenyl,alkynyl or cycloalkyl substituents with one or more of any of O, S, SO,SO₂, N, or N-alkyl, including, for example, —CH₂—O—CH₂, —CH₂—SO₂—CH₂,—CH₂—NH—CH₃ and so forth.

The term “oxo” as used herein refers to the substituent ═O.

The term “halogen” or “halo” as used herein includes iodine, bromine,chlorine and fluorine.

The term “mercapto” as used herein refers to the substituent —SH.

The term “carboxy” as used herein refers to the substituent —COOH.

The term “cyano” as used herein refers to the substituent —CN.

The term “aminosulfonyl” as used herein refers to the substituent—SO₂NH₂.

The term “carbamoyl” as used herein refers to the substituent —C(O)NH₂.

The term “sulfanyl” as used herein refers to the substituent —S—.

The term “sulfenyl” as used herein refers to the substituent —S(O)—.

The term “sulfonyl” as used herein refers to the substituent —S(O)₂—.

The term “ethoxy” as used herein refers to the substituent —O—CH₂CH₃.

The term “methoxy” as used herein refers to the substituent —O—CH₃.

As used herein, the term “optionally” means that the subsequentlydescribed event(s) may or may not occur, and includes both event(s)which occur and events that do not occur.

Compounds of structural formula I and formulas Ia-f may contain one ormore asymmetric centers and can thus occur as racemates and racemicmixtures, single enantiomers, diastereomeric mixtures and individualdiastereomers. The present invention is meant to comprehend all suchisomeric forms of the compounds of structural formula I and formulasIa-f.

Compounds of structural formula I and formulas Ia-f may be separatedinto their individual diastereoisomers by, for example, fractionalcrystallization from a suitable solvent, for example methanol or ethylacetate or a mixture thereof, or via chiral chromatography using anoptically active stationary phase. Absolute stereochemistry may bedetermined by X-ray crystallography of crystalline products orcrystalline intermediates which are derivatized, if necessary, with areagent containing an asymmetric center of known absolute configuration.

Alternatively, any stereoisomer of a compound of the general structuralformula I and formulas Ia-f may be obtained by stereospecific synthesisusing optically pure starting materials or reagents of known absoluteconfiguration.

If desired, racemic mixtures of the compounds may be separated so thatthe individual enantiomers are isolated. The separation can be carriedout by methods well known in the art, such as the coupling of a racemicmixture of compounds to an enantiomerically pure compound to form adiastereomeric mixture, followed by separation of the individualdiastereomers by standard methods, such as fractional crystallization orchromatography. The coupling reaction is often the formation of saltsusing an enantiomerically pure acid or base. The diasteromericderivatives may then be converted to the pure enantiomers by cleavage ofthe added chiral residue. The racemic mixture of the compounds can alsobe separated directly by chromatographic methods utilizing chiralstationary phases, which methods are well known in the art.

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and Zgeometric isomers.

Some of the compounds described herein may exist as tautomers, whichhave different points of attachment of hydrogen accompanied by one ormore double bond shifts. For example, a ketone and its enol form areketo-enol tautomers. The individual tautomers as well as mixturesthereof are encompassed with compounds of the present invention.

In the compounds of generic Formula I and formulas Ia-f, the atoms mayexhibit their natural isotopic abundances, or one or more of the atomsmay be artificially enriched in a particular isotope having the sameatomic number, but an atomic mass or mass number different from theatomic mass or mass number predominantly found in nature. The presentinvention is meant to include all suitable isotopic variations of thecompounds of generic Formula I and formulas Ia-g. For example, differentisotopic forms of hydrogen (H) include protium (1H) and deuterium (2H).Protium is the predominant hydrogen isotope found in nature. Enrichingfor deuterium may afford certain therapeutic advantages, such asincreasing in vivo half-life or reducing dosage requirements, or mayprovide a compound useful as a standard for characterization ofbiological samples. Isotopically-enriched compounds within genericFormula I and formulas Ia-f can be prepared without undueexperimentation by conventional techniques well known to those skilledin the art or by processes analogous to those described in the Schemesand Examples herein using appropriate isotopically-enriched reagentsand/or intermediates.

It will be understood that, as used herein, references to the compoundsof structural formula I and formulas Ia-f are meant to also include thepharmaceutically acceptable salts, and also salts that are notpharmaceutically acceptable when they are used as precursors to the freecompounds or their pharmaceutically acceptable salts or in othersynthetic manipulations.

Compositions

According to another aspect, the described invention providespharmaceutical compositions comprising a therapeutic amount of at leastone of the small molecule anti-cancer compounds and a pharmaceuticallyacceptable carrier.

The term “active” as used herein refers to having pharmacological orbiological activity or affect. The term “active ingredient” (“AI”,“active pharmaceutical ingredient”, or “bulk active”) is the substancein a drug that is pharmaceutically active.

The terms “formulation” and “composition” are used interchangeablyherein to refer to a product of the described invention that comprisesall active and inert ingredients. The terms “pharmaceutical formulation”or “pharmaceutical composition” as used herein refer to a formulation orcomposition that is employed to prevent, reduce in intensity, cure orotherwise treat a target condition or disease.

As used herein, the term “binder” refers to substances that bind or“glue” powders together and make them cohesive by forming granules, thusserving as the “adhesive” in the formulation. Binders add cohesivestrength already available in the diluent or bulking agent. Exemplarybinders include sugars such as sucrose; starches derived from wheat,corn rice and potato; natural gums such as acacia, gelatin andtragacanth; derivatives of seaweed such as alginic acid, sodium alginateand ammonium calcium alginate; cellulosic materials such asmethylcellulose and sodium carboxymethylcellulose andhydroxypropylmethylcellulose; polyvinylpyrrolidone; and inorganics suchas magnesium aluminum silicate. The amount of binder in the compositioncan range from about 2 to about 20% by weight of the composition, morepreferably from about 3 to about 10% by weight, even more preferablyfrom about 3 to about 6% by weight.

As used herein, the term “bioavailability” refers to the rate and extentto which the active drug ingredient or therapeutic moiety is absorbedinto the systemic circulation from an administered dosage form ascompared to a standard or control.

As used herein, the term “capsule” refers to a special container orenclosure made of methyl cellulose, polyvinyl alcohols, or denaturedgelatins or starch for holding or containing compositions comprising theactive ingredients. Hard shell capsules are typically made of blends ofrelatively high gel strength bone and pork skin gelatins. The capsuleitself may contain small amounts of dyes, opaquing agents, plasticizersand preservatives.

As used herein, the term “coloring agents” refers to excipients thatprovide coloration to the composition or the dosage form. Suchexcipients can include food grade dyes and food grade dyes adsorbed ontoa Exemplary adsorbent such as clay or aluminum oxide. The amount of thecoloring agent can vary from about 0.1 to about 5% by weight of thecomposition, preferably from about 0.1 to about 1%.

As used herein, the term “diluent” refers to substances that usuallymake up the major portion of the composition or dosage form. Exemplarydiluents include sugars such as lactose, sucrose, mannitol and sorbitol;starches derived from wheat, corn, rice and potato; and celluloses suchas microcrystalline cellulose. The amount of diluent in the compositioncan range from about 10 to about 90% by weight of the total composition,preferably from about 25 to about 75%, more preferably from about 30 toabout 60% by weight, even more preferably from about 12 to about 60%.

As used herein, the term “disintegrant” refers to materials added to thecomposition to help it break apart (disintegrate) and release themedicaments. Exemplary disintegrants include starches; “cold watersoluble” modified starches such as sodium carboxymethyl starch; naturaland synthetic gums such as locust bean, karaya, guar, tragacanth andagar; cellulose derivatives such as methylcellulose and sodiumcarboxymethylcellulose; microcrystalline celluloses and cross-linkedmicrocrystalline celluloses such as sodium croscarmellose; alginatessuch as alginic acid and sodium alginate; clays such as bentonites; andeffervescent mixtures. The amount of disintegrant in the composition canrange from about 2 to about 15% by weight of the composition, morepreferably from about 4 to about 10% by weight.

As used herein, the term “glident” refers to material that preventscaking and improves the flow characteristics of granulations, so thatflow is smooth and uniform. Exemplary glidents include silicon dioxideand talc. The amount of glident in the composition can range from about0.1% to about 5% by weight of the total composition, preferably fromabout 0.5 to about 2% by weight.

As used herein, the term “lubricant” refers to a substance added to thedosage form to enable the tablet, granules, etc. after it has beencompressed, to release from the mold or die by reducing friction orwear. Exemplary lubricants include metallic stearates such as magnesiumstearate, calcium stearate or potassium stearate; stearic acid; highmelting point waxes; and water soluble lubricants such as sodiumchloride, sodium benzoate, sodium acetate, sodium oleate, polyethyleneglycols and d'l-leucine. Lubricants are usually added at the very laststep before compression, since they must be present on the surfaces ofthe granules and in between them and the parts of the tablet press. Theamount of lubricant in the composition can range from about 0.2 to about5% by weight of the composition, preferably from about 0.5 to about 2%,more preferably from about 0.3 to about 1.5% by weight.

As used herein, the term “oral gel” refers to the active ingredientsdispersed or solubilized in a hydrophillic semi-solid matrix.

As used herein, the term “tablet” refers to a compressed or molded soliddosage form containing the active ingredients with suitable diluents.The tablet can be prepared by compression of mixtures or granulationsobtained by wet granulation, dry granulation or by compaction.

As used herein, the term “therapeutic amount” refers to the amount of asmall molecule anti-cancer compound of the described invention that iseffective to modulate a cancer cell sensitive to cholesterolbiosynthesis pathway inhibition. Combined with the teachings providedherein, by choosing among the various active compounds and weighingfactors such as potency, relative bioavailability, patient body weight,severity of adverse side-effects and preferred mode of administration,an effective prophylactic or therapeutic treatment regimen may beplanned which does not cause substantial toxicity and yet is effectiveto treat the particular subject. The effective amount for any particularapplication may vary depending on such factors as the disease orcondition being treated, the particular described compound, the size ofthe subject, or the severity of the disease or condition. One ofordinary skill in the art may determine empirically the therapeuticallyeffective amount of a particular described compound and/or othertherapeutic agent without necessitating undue experimentation. It isgenerally preferred that a maximum dose be used, that is, the highestsafe dose according to some medical judgment. The terms “dose” and“dosage” are used interchangeably herein.

For any compound described herein the therapeutically effective amountcan be initially determined from preliminary in vitro studies and/oranimal models. A therapeutically effective dose can also be determinedfrom human data. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound.

The formulations of inhibitors may be administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeutic agents.

According to another embodiment, the compositions of the describedinvention can further include one or more additional compatible activeingredients. “Compatible” as used herein means that the components ofsuch a composition are capable of being combined with each other in amanner such that there is no interaction that would substantially reducethe efficacy of the composition under ordinary use conditions. As usedherein, the phrase “additional active ingredient” refers to an agent,other than a small molecule anticancer compound of the describedcomposition, that exerts a pharmacological, or any other beneficialactivity. Nonlimiting examples of such additional therapeutic agentsinclude, without limitation, 5-fluorouracil, leucovorin, oxaliplatincapecitabine, leucovorin, irinotecan, capecitabine, oxaliplatin,bevacizumab, cetuximab, panitumumab, or a combination thereof.

Pharmaceutically Acceptable Carrier

The term “pharmaceutically-acceptable carrier” as used herein refers toone or more compatible solid or liquid filler, diluents or encapsulatingsubstances which are Exemplary for administration to a human or othervertebrate animal. The term “carrier” as used herein refers to anorganic or inorganic ingredient, natural or synthetic, with which theactive ingredient is combined to facilitate the application. Accordingto some embodiments, the carrier can be inert, or it can possesspharmaceutical benefits.

The components of the pharmaceutical compositions also are capable ofbeing commingled in a manner such that there is no interaction whichwould substantially impair the desired pharmaceutical efficiency.

The carrier can be liquid or solid and is selected with the plannedmanner of administration in mind to provide for the desired bulk,consistency, etc., when combined with an active and the other componentsof a given composition.

Administration

For use in therapy, a therapeutic amount of a small molecule anticancercompound may be administered to a subject by any mode. Administering thepharmaceutical composition may be accomplished by any means known to theskilled artisan. Routes of administration include, but are not limitedto, parenteral oral, buccal, topical, by inhalation or insufflation(i.e., through the mouth or through the nose), or rectal.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle); intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord), intrasternal injection, or infusiontechniques. A parenterally administered composition of the presentinvention is delivered using a needle, e.g., a surgical needle. The term“surgical needle” as used herein, refers to any needle adapted fordelivery of fluid (i.e., capable of flow) compositions of the presentinvention into a selected anatomical structure. Injectable preparations,such as sterile injectable aqueous or oleaginous suspensions, may beformulated according to the known art using Exemplary dispersing orwetting agents and suspending agents.

The compositions of the present invention may be in the form of asterile injectable aqueous solution or oleaginous suspension. A solutiongenerally is considered as a homogeneous mixture of two or moresubstances; it is frequently, though not necessarily, a liquid. In asolution, the molecules of the solute (or dissolved substance) areuniformly distributed among those of the solvent. A suspension is adispersion in which a finely-divided species is combined with anotherspecies, with the former being so finely divided and mixed that itdoesn't rapidly settle out. The term “dispersion”, as used herein,refers to a two-phase system, in which one phase is distributed asparticles or droplets in the second, or continuous phase. In thesesystems, the dispersed phase frequently is referred to as thediscontinuous or internal phase, and the continuous phase is called theexternal phase or dispersion medium. For example, in coarse dispersions,the particle size is 0.5 mm. In colloidal dispersions, size of thedispersed particle is in the range of approximately 1 nm to 0.5 mm.Molecular dispersion is a dispersion, in which the dispersed phaseconsists of individual molecules; if the molecules are less thancolloidal size, the result is a true solution.

The compositions of the described invention also may be in the form ofan emulsion. An emulsion is a two-phase system prepared by combining twoimmiscible liquid carriers, one of which is disbursed uniformlythroughout the other and consists of globules that have diameters equalto or greater than those of the largest colloidal particles. The globulesize is critical and must be such that the system achieves maximumstability. Usually, separation of the two phases will not occur unless athird substance, an emulsifying agent, is incorporated. Thus, a basicemulsion contains at least three components, the two immiscible liquidcarriers and the emulsifying agent, as well as the active ingredient.Most emulsions incorporate an aqueous phase into a non-aqueous phase (orvice versa). However, it is possible to prepare emulsions that arebasically non-aqueous, for example, anionic and cationic surfactants ofthe non-aqueous immiscible system glycerin and olive oil. Thus, thecompositions of the invention may be in the form of an oil-in-wateremulsion. The oily phase may be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example a liquid paraffin, or amixture thereof. Exemplary emulsifying agents may be naturally-occurringgums, for example, gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of the partial esterswith ethylene oxide, for example, polyoxyethylene sorbitan monooleate.

According to some embodiments, the composition may be formulated forparenteral administration by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form. Additionally, suspensions ofthe active compounds may be prepared as appropriate oily injectionsuspensions. Exemplary lipophilic solvents or vehicles include fattyoils such as sesame oil, or synthetic fatty acid esters, such as ethyloleate or triglycerides, or liposomes. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension also may contain Exemplary stabilizers or agents, whichincrease the solubility of the compounds to allow for the preparation ofhighly concentrated solutions. Alternatively, the active compounds maybe in powder form for constitution with a Exemplary vehicle, e.g.,sterile pyrogen-free water, before use.

The pharmaceutical compositions also may comprise Exemplary solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Exemplary liquid or solid pharmaceutical preparation forms are, forexample, microencapsulated, and if appropriate, with one or moreexcipients, encochleated, coated onto microscopic gold particles,contained in liposomes, pellets for implantation into the tissue, ordried onto an object to be rubbed into the tissue. Such pharmaceuticalcompositions also may be in the form of granules, beads, powders,tablets, coated tablets, (micro)capsules, suppositories, syrups,emulsions, suspensions, creams, drops or preparations with protractedrelease of active compounds, in whose preparation excipients andadditives and/or auxiliaries such as disintegrants, binders, coatingagents, swelling agents, lubricants, or solubilizers are customarilyused as described above. The pharmaceutical compositions are Exemplaryfor use in a variety of drug delivery systems. For a brief review ofmethods for drug delivery, see Langer 1990 Science 249, 1527-1533, whichis incorporated herein by reference.

Depending upon the structure, a therapeutic amount of at least one smallmolecule anti-cancer compound effective to modulate a cancer cellsensitive to cholesterol biosynthesis inhibition of the describedinvention, and optionally at least one additional active agent, may beadministered per se (neat) or, depending upon the structure of theinhibitor, in the form of a pharmaceutically acceptable salt. Theinhibitors of the described invention may form pharmaceuticallyacceptable salts with organic or inorganic acids, or organic orinorganic bases. When used in medicine the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsconveniently may be used to prepare pharmaceutically acceptable saltsthereof. Such salts include, but are not limited to, those prepared fromthe following acids: hydrochloric, hydrobromic, sulphuric, nitric,phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric,citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts may beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

By “pharmaceutically acceptable salt” is meant those salts which are,within the scope of sound medical judgment, Exemplary for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell-known in the art. For example, P. H. Stahl, et al. describepharmaceutically acceptable salts in detail in “Handbook ofPharmaceutical Salts: Properties, Selection, and Use” (Wiley VCH,Zurich, Switzerland: 2002).

The salts may be prepared in situ during the final isolation andpurification of the compounds described within the described inventionor separately by reacting a free base function with a Exemplary organicacid. Representative acid addition salts include, but are not limitedto, acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate(isethionate), lactate, maleate,methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate and undecanoate. Also, the basicnitrogen-containing groups may be quaternized with such agents as loweralkyl halides, such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyland diamyl sulfates; long chain halides, such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides; arylalkyl halides, such asbenzyl and phenethyl bromides, and others. Water or oil-soluble ordispersible products are thereby obtained. Examples of acids which maybe employed to form pharmaceutically acceptable acid addition saltsinclude such inorganic acids as hydrochloric acid, hydrobromic acid,sulphuric acid and phosphoric acid and such organic acids as oxalicacid, maleic acid, succinic acid and citric acid. Basic addition saltsmay be prepared in situ during the final isolation and purification ofcompounds described within the invention by reacting a carboxylicacid-containing moiety with an Exemplary base such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium and aluminum salts and the likeand nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, diethylamine, ethylamine and the like.Other representative organic amines useful for the formation of baseaddition salts include ethylenediamine, ethanolamine, diethanolamine,piperidine, piperazine and the like. Pharmaceutically acceptable saltsmay be also obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound such as an aminewith a Exemplary acid affording a physiologically acceptable anion.Alkali metal (for example, sodium, potassium or lithium) or alkalineearth metal (for example calcium or magnesium) salts of carboxylic acidsmay also be made.

The formulations may be presented conveniently in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing into association a composition,or a pharmaceutically acceptable salt or solvate thereof (“activecompound”) with the carrier which constitutes one or more accessoryagents. In general, the formulations are prepared by uniformly andintimately bringing into association the active agent with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

The pharmaceutical agent or a pharmaceutically acceptable ester, salt,solvate or prodrug thereof may be mixed with other active materials thatdo not impair the desired action, or with materials that supplement thedesired action. Solutions or suspensions used for parenteral,intradermal, subcutaneous, intrathecal, or topical application mayinclude, but are not limited to, for example, the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationmay be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic. Administered intravenously, particularcarriers are physiological saline or phosphate buffered saline (PBS).

Pharmaceutical compositions for parenteral injection comprisepharmaceutically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of Exemplary aqueous and nonaqueous carriers, diluents,solvents or vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), Exemplary mixturesthereof, vegetable oils (such as olive oil) and injectable organicesters such as ethyl oleate. Proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions, and by the use ofsurfactants.

These compositions also may contain adjuvants including preservativeagents, wetting agents, emulsifying agents, and dispersing agents.Prevention of the action of microorganisms may be ensured by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. It also may bedesirable to include isotonic agents, for example, sugars, sodiumchloride and the like. Prolonged absorption of the injectablepharmaceutical form may be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Suspensions, in addition to the active compounds, may contain suspendingagents, as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, andmixtures thereof.

The therapeutic agent(s), including the composition(s) of the describedinvention may be provided in particles. The term “particles” as usedherein refers to nano or microparticles (or in some instances larger)that may contain in whole or in part the composition or the othertherapeutic agent(s) as described herein. The particles may contain thetherapeutic agent(s) in a core surrounded by a coating. The therapeuticagent(s) also may be dispersed throughout the particles. The therapeuticagent(s) also may be adsorbed into the particles. The particles may beof any order release kinetics, including zero order release, first orderrelease, second order release, delayed release, sustained release,immediate release, etc., and any combination thereof. The particle mayinclude, in addition to the therapeutic agent(s), any of those materialsroutinely used in the art of pharmacy and medicine, including, but notlimited to, erodible, nonerodible, biodegradable, or nonbiodegradablematerial or combinations thereof. The particles may be microcapsulesthat contain the composition in a solution or in a semi-solid state. Theparticles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials may be usedin the manufacture of particles for delivering the therapeutic agent(s).Such polymers may be natural or synthetic polymers. The polymer isselected based on the period of time over which release is desired. Forexample, bioadhesive polymers include bioerodible hydrogels as describedby Sawhney et al in Macromolecules (1993) 26, 581-587, the teachings ofwhich are incorporated herein by reference. These include polyhyaluronicacids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid,alginate, chitosan, poly(methyl methacrylates), poly(ethylmethacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate).

The therapeutic agent(s) may be contained in controlled release systems.In order to prolong the effect of a drug, it often is desirable to slowthe absorption of the drug from subcutaneous, intrathecal, orintramuscular injection. This may be accomplished by the use of a liquidsuspension of crystalline or amorphous material with poor watersolubility. The rate of absorption of the drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. The term “controlled release” is intended to refer toany drug-containing formulation in which the manner and profile of drugrelease from the formulation are controlled. This refers to immediate aswell as non-immediate release formulations, with non-immediate releaseformulations including, but not limited to, sustained release anddelayed release formulations. The term “sustained release” (alsoreferred to as “extended release”) is used herein in its conventionalsense to refer to a drug formulation that provides for gradual releaseof a drug over an extended period of time, and that can result insubstantially constant blood levels of a drug over an extended timeperiod. Alternatively, delayed absorption of a parenterally administereddrug form is accomplished by dissolving or suspending the drug in an oilvehicle. The term “delayed release” is used herein in its conventionalsense to refer to a drug formulation in which there is a time delaybetween administration of the formulation and the release of the drugthere from. “Delayed release” may or may not involve gradual release ofdrug over an extended period of time, and thus may or may not be“sustained release.”

According to some embodiments, use of a long-term sustained releaseimplant may be desirable for treatment of chronic conditions. The term“long-term” release, as used herein, means that the implant isconstructed and arranged to deliver therapeutic levels of the activeingredient for at least 7 days, and preferably about 30 to about 60days. Long-term sustained release implants are well-known to those ofordinary skill in the art and include some of the release systemsdescribed above.

Injectable depot forms are made by forming microencapsulated matrices ofa described inhibitor in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of inhibitor topolymer and the nature of the particular polymer employed, the rate ofdrug release may be controlled. Such long acting formulations may beformulated with appropriate polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Examples of other biodegradable polymers include poly(orthoesters)and poly(anhydrides). Depot injectable formulations also are prepared byentrapping the inhibitor of the described invention in liposomes ormicroemulsions, which are compatible with body tissues.

The injectable formulations may be sterilized, for example, byfiltration through a bacterial-retaining filter or by incorporatingsterilizing agents in the form of sterile solid compositions that may bedissolved or dispersed in sterile water or other sterile injectablemedium just prior to use. Injectable preparations, for example, sterileinjectable aqueous or oleaginous suspensions may be formulated accordingto the known art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation also may be asterile injectable solution, suspension or emulsion in a nontoxic,parenterally acceptable diluent or solvent such as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P. and isotonic sodiumchloride solution. In addition, sterile, fixed oils conventionally areemployed or as a solvent or suspending medium. For this purpose anybland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid are used inthe preparation of injectables.

Formulations for parenteral (including but not limited to, subcutaneous,intradermal, intramuscular, intravenous, intrathecal and intraarticular)administration include aqueous and non-aqueous sterile injectionsolutions that may contain anti-oxidants, buffers, bacteriostats andsolutes, which render the formulation isotonic with the blood of theintended recipient; and aqueous and non-aqueous sterile suspensions,which may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials, and may be stored in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline, water-for-injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindpreviously described.

Exemplary buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Exemplary preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

For oral administration in the form of tablets or capsules, the activedrug component may be combined with any oral non-toxic pharmaceuticallyacceptable inert carrier, such as lactose, starch, sucrose, cellulose,magnesium stearate, dicalcium phosphate, calcium sulfate, talc,mannitol, ethyl alcohol (liquid forms) and the like. Moreover, whendesired or needed, suitable binders, lubricants, disintegrating agentsand coloring agents also may be incorporated in the mixture. Powders andtablets may comprise from about 5 to about 95 percent of the describedcomposition. Exemplary binders include starch, gelatin, natural sugars,corn sweeteners, natural and synthetic gums such as acacia, sodiumalginate, carboxymethylcellulose, polyethylene glycol and waxes. Amongthe lubricants there may be mentioned for use in these dosage forms,boric acid, sodium benzoate, sodium acetate, sodium chloride, and thelike. Disintegrants include starch, methylcellulose, guar gum and thelike. Sweetening and flavoring agents and preservatives may also beincluded where appropriate.

The compositions of the invention also may be formulated as syrups andelixirs. Syrups and elixirs may be formulated with sweetening agents,for example, glycerol, propylene glycol, sorbitol or sucrose. Suchformulations also may contain a demulcent, a preservative, and flavoringand coloring agents. Demulcents are protective agents employed primarilyto alleviate irritation, particularly mucous membranes or abradedtissues. A number of chemical substances possess demulcent properties.These substances include the alginates, mucilages, gums, dextrins,starches, certain sugars, and polymeric polyhydric glycols. Othersinclude acacia, agar, benzoin, carbomer, gelatin, glycerin, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,propylene glycol, sodium alginate, tragacanth, hydrogels and the like.

For buccal administration, the compositions of the present invention maytake the form of tablets or lozenges formulated in a conventional mannerfor this route.

Liquid form preparations include solutions, suspensions and emulsions.

Liquid form preparations also may include solutions for intranasaladministration.

The compositions of the present invention may be in the form of adispersible dry powder for delivery by inhalation or insufflation(either through the mouth or through the nose). Dry powder compositionsmay be prepared by processes known in the art, such as lyophilizationand jet milling, as disclosed in International Patent Publication No. WO91/16038 and as disclosed in U.S. Pat. No. 6,921,527, the disclosures ofwhich are incorporated by reference. The composition of the presentinvention is placed within a Exemplary dosage receptacle in an amountsufficient to provide a subject with a unit dosage treatment. The dosagereceptacle is one that fits within a Exemplary inhalation device toallow for the aerosolization of the dry powder composition by dispersioninto a gas stream to form an aerosol and then capturing the aerosol soproduced in a chamber having a mouthpiece attached for subsequentinhalation by a subject in need of treatment. Such a dosage receptacleincludes any container enclosing the composition known in the art suchas gelatin or plastic capsules with a removable portion that allows astream of gas (e.g., air) to be directed into the container to dispersethe dry powder composition. Such containers are exemplified by thoseshown in U.S. Pat. No. 4,227,522; U.S. Pat. No. 4,192,309; and U.S. Pat.No. 4,105,027. Exemplary containers also include those used inconjunction with Glaxo's Ventolin® Rotohaler brand powder inhaler orFison's Spinhaler® brand powder inhaler. Another Exemplary unit-dosecontainer which provides a superior moisture barrier is formed from analuminum foil plastic laminate. The pharmaceutical-based powder isfilled by weight or by volume into the depression in the formable foiland hermetically sealed with a covering foil-plastic laminate. Such acontainer for use with a powder inhalation device is described in U.S.Pat. No. 4,778,054 and is used with Glaxo's Diskhaler® (U.S. Pat. Nos.4,627,432; 4,811,731; and 5,035,237). Each of these references isincorporated herein by reference.

The compositions of the present invention may be in the form ofsuppositories for rectal administration of the composition. “Rectal” or“rectally” as used herein refers to introduction into the body throughthe rectum where absorption occurs through the walls of the rectum.These compositions can be prepared by mixing the drug with a Exemplarynonirritating excipient such as cocoa butter and polyethylene glycolswhich are solid at ordinary temperatures but liquid at the rectaltemperature and will therefore melt in the rectum and release the drug.When formulated as a suppository the compositions of the invention maybe formulated with traditional binders and carriers, such astriglycerides.

The term “topical” refers to administration of an inventive compositionat, or immediately beneath, the point of application. The phrase“topically applying” describes application onto one or more surfaces(s)including epithelial surfaces. Although topical administration, incontrast to transdermal administration, generally provides a localrather than a systemic effect, as used herein, unless otherwise statedor implied, the terms topical administration and transdermaladministration are used interchangeably. For the purpose of thisapplication, topical applications shall include mouthwashes and gargles.

Topical administration may also involve the use of transdermaladministration such as transdermal patches or iontophoresis deviceswhich are prepared according to techniques and procedures well known inthe art. The terms “transdermal delivery system”, transdermal patch” or“patch” refer to an adhesive system placed on the skin to deliver a timereleased dose of a drug(s) by passage from the dosage form through theskin to be available for distribution via the systemic circulation.Transdermal patches are a well-accepted technology used to deliver awide variety of pharmaceuticals, including, but not limited to,scopolamine for motion sickness, nitroglycerin for treatment of anginapectoris, clonidine for hypertension, estradiol for post-menopausalindications, and nicotine for smoking cessation.

Patches exemplary for use in the present invention include, but are notlimited to, (1) the matrix patch; (2) the reservoir patch; (3) themulti-laminate drug-in-adhesive patch; and (4) the monolithicdrug-in-adhesive patch; TRANSDERMAL AND TOPICAL DRUG DELIVERY SYSTEMS,pp. 249-297 (Tapash K. Ghosh et al. eds., 1997), hereby incorporatedherein by reference. These patches are well known in the art andgenerally available commercially.

According to some embodiments, the solid tumor is a brain tumor.According to some embodiments the brain tumor is a glioma. According tosome embodiments, the glioma is a glioblastoma multiforme. According tosome embodiments, the solid brain tumor comprises cancer stem cells.According to some embodiments, the therapeutic amount of the smallmolecule anti-cancer compound is effective to inhibit tumor growth,inhibit tumor proliferation, induce cell death, or a combinationthereof. According to some embodiments, the therapeutic amount of thesmall molecule anti-cancer compounds is effective to selectively inhibitgrowth of the cancer stem cells, proliferation of the cancer stem cells,to induce cell death of the cancer stem cells, or a combination thereof,without affecting normally dividing cells. According to someembodiments, the therapeutic amount is effective to inhibit acholesterol biosynthesis pathway.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein also can beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural references unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the described inventionis not entitled to antedate such publication by virtue of priorinvention. Further, the dates of publication provided may be differentfrom the actual publication dates which may need to be independentlyconfirmed.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1. Compounds

A mouse colony with 100% incidence of GBM and with conditional knockoutof p53, pTEN and NF1 genes (Mut6; glioblastoma) was used to isolateprimary neuronal stem cells from GBM tumors cultured in 5% Oxygen and inserum free media with defined growth factors. Primary cells frommultiple tumors were pooled for high throughput screening with controlsagainst the University of Texas Southwestern (UTSW) compound file, whichencompasses ˜200,000 synthetic compounds that represent a large chemicalspace from several commercial vendors, including 1200 marketed drugsfrom the Prestwick Chemical Library®, and 600 compounds that went topre-clinical tests from the NIH library.

A luminescence-based Celltiter-Glo® assay was performed to measure cellviability, using ATP levels as the readout. In brief, opaque-walledmultiwell plates with Mut6 cells in culture medium (250 per well,384-well plates) were prepared. Control wells containing medium withoutcells were prepared to obtain a value for background luminescence. Testcompounds were added to experimental wells, and incubated according toculture protocol. The plate and its contents were incubated at 37° C.,5% Oxygen for 96 hours. An ATP standard curve was generated immediatelyprior to adding the CellTiter-Glo® Reagent. A volume of CellTiter-Glo®Reagent equal to the volume of cell culture medium present in each well(25 μl of reagent to 25 μl of medium containing cells for a 384-wellplate) was added. The contents were mixed for 2 minutes on an orbitalshaker to induce cell lysis. The plate was allowed to incubate at roomtemperature for 10 minutes to stabilize the luminescent signal andluminescence recorded. (e.g. GloMax®, Lumistar, SPECTROstar, PHERAstarFS).

This primary screen yielded 4480 positive hits with ≥50% inhibition ofcell consumption (based on a z-score of ≤−3, which means that thez-score of −3 was 3 standard deviations below the mean). The compoundswere further screened to identify compounds that displayed ≥70%inhibition of Mut6 ATP consumption. This screen identified 1078compounds. The compounds were tested against normally dividing cells,e.g., wild type mouse embryonic fibroblasts, wild type mouse astrocytes,and wild type mouse SVZ stem cells. Any compound toxic to all three wildtype normally dividing cells was removed, resulting in 713 smallmolecules, of which, after counterscreening at lower molarity againstnormal astrocytes and mouse embryonic fibroblasts (MEFs), 61 compoundswere determined to kill only the cancer stem cells. The 61 compoundswere analyzed by S9 fraction assay and hepatocyte assay, leaving 17candidate compounds. The overall screening strategy is shown in FIG. 6.

S9 Metabolism Assay

Female ICR/CD-1 mouse S9 fractions were purchased from Celsis/In VitroTechnologies (Baltimore, Md.). 25 μl (0.5 mg) of S9 protein was added toa 15 ml glass screw cap tube. 350 μl of a 50 mM Tris, pH 7.5 solution,containing the compound of interest was added on ice. The finalconcentration of compound after addition of all reagents was 2 μM. 125μl of an NADPH-regenerating system (1.7 mg/ml NADP, 7.8 mg/mlglucose-6-phosphate, 6 U/ml glucose-6-phosphate dehydrogenase in 2% w/vNaHCO₃/10 mm MgCl₂) was added for analysis of Phase I metabolism.Uridine 5′-diphospho-α-D-glucuronic acid (UDPGA 1.9 mg/ml) and3′-phosphoadenosine-5′-phosphosulphate (PAPS, 100 mg/ml) wereadditionally added for phase II reactions. The tube was then placed in a37° C. shaking water bath. At varying time points after addition ofphase I and phase II cofactors, the reaction was stopped by the additionof 0.5 ml of methanol containing 0.2% formic acid and 200 ng/ml internalstandard (either n-benzylbenzamide or tolbutamide, Sigma, St. Louis,Mo.). The samples were incubated 10′ at RT and then spun at 16,100×g for5 min in a microcentrifuge. The supernatant was analyzed by LC-MS/MS.Metabolism of 7-ethoxycoumarin was used to monitor hepatocyteperformance. Analytical methods were developed for each compound usingan Applied Biosystems (Foster City, Calif.) 4000-QTrap, a combinationtriple quadrupole/ion trap instrument. The parent ion and the two mostprominent daughter ions were followed to confirm compound identity,although only the most abundant daughter was used for quantitation. AShimadzu (Columbia, Md.) Prominence LC with Agilent C18 XDB column (5micron packing; 50×4.6 mm) was used for chromatography.

Hepatocyte Metabolism Assay

Male ICR/CD-1 mouse hepatocytes, InVitroGRO HI and HT Medium, and CelsisTorpedo Antibiotic Mix were purchased from Celsis/In Vitro Technologies(Baltimore, Md.). Cryopreserved hepatocytes were thawed in HT Mediacontaining antibiotics, resuspended in HI media at 2×106/mL and platedin 96-well plates at 0.05 mL (10⁵ cells)/well. Compounds to be testedwere dissolved in DMSO at 2 mM, further diluted to 4 μM in HI media, andadded to the cells in 50 μL so that the final compound concentration was2 μM. Two additional wells containing compound and no cells were platedto serve as time 0 (C₀) and end-point solvent control (Cep). The cellswere then placed in a 37° C., 5% CO₂ incubator. At the time pointsindicated, the well contents were harvested and a 2-fold volume ofmethanol containing 0.15% formic acid and 150 ng/ml internal standard(either n-benzylbenzamide or tolbutamide, Sigma, St. Louis, Mo.) addedto lyse the cells and precipitate proteins. The samples were incubated10 min at RT and then spun at 16,100 g for 5 min in a microcentrifuge.The supernatant was analyzed by LC-MS/MS. Metabolism of 7-ethoxycoumarinwas used to monitor hepatocyte performance. Analytical methods weredeveloped for each compound using an Applied Biosystems (Foster City,Calif.) 4000-QTrap, a combination triple quadrupole/ion trap instrument.The parent ion and the two most prominent daughter ions were followed toconfirm compound identity, although only the most abundant daughter wasused for quantitation. A Shimadzu (Columbia, Md.) Prominence LC withAgilent C18 XDB column (5 micron packing; 50×4.6 mm) was used forchromatography.

Example 2. Candidate Compound DS-1-033 (Hereinafter Compound 4C12)Showed Selective Toxicity Towards Mut6 Cells

FIG. 7 shows that compound 4C12 induces cell death in Mut6 tumor cells.Normal astrocytes, Mut6 cells and mouse embryonic fibroblasts weretreated with increasing concentrations of compound 4C12. FIG. 7A, whichshows a plot of relative ATP activity (y axis, a measure of viability)versus concentration (nM), shows that compound 4C12 has an ED50 of 50 nMagainst Mut6 cells. Normal astrocytes and MEFs were unaffected bycompound 4C12. FIG. 7B shows phase contrast microscopy of Mut6 cells andcontrol MEFs after 14 hours, 23 hours and 38 hours treatment withcompound 4C12 and a negative control containing vehicle only. The plotbelow shows that all cells treated with vehicle (negative control)remained viable, while only 50% of the Mut6 tumor cells treated withcompound 4C12 were viable after 96 hours.

Compound 4C12 served as initial lead compound for analog development andfor additional studies. Exemplary analog compounds and their propertiesare shown in Table A. A druglikeness central nervous systemmultiparameter optimization (CNS MPO) algorithm was built using a set ofsix physicochemical parameters, i.e., [(a) lipophilicity, calculatedpartition coefficient (ClogP); (b) calculated distribution coefficientat pH=7.4 (ClogD); (c) molecular weight (MW); (d) topological polarsurface area (TPSA); (e) number of hydrogen bond donors (HBD); (f) mostbasic center (pK(a))). See Wager, T T et al, “Moving beyond rules: thedevelopment of a central nervous system multi-parameter-optimization(CNS MPO) approach to enable alignment of drug-like properties,” ACSChem. Neurosci. 1(6): 435-49 (2010). IC50 curves for the analogcompounds plotted as ATP activity (y axis, a measure of viability) vs.concentration (nM) (x-axis) are shown in FIG. 9.

General Procedure A-1:

In a flask equipped with a reflux condenser, the carboxylic acid (10mmol, 1 eq) and sodium acetate (1.1 eq) were suspended in water (20 mL).4-Fluorothiophenol (1.1 eq) was introduced in one portion and thereaction mixture was stirred at 90° C. After 4-16 h, the resulting solidproduct was filtered at room temperature and washed with water (80 mL)and hexanes (30 mL), and dried under high vacuum.

General Procedure A-2:

A 15 mL pressure tube was charged with the aryl fluoride DS-1-095 (0.5mmol, 1 eq), water (2 mL), the nucleophile (1.1 eq) and sodiumbicarbonate (1.1 eq). The tube was sealed and the reaction mixture wasstirred at 90° C. for 2 h. A precipitate appeared and the suspension wasstirred overnight at room temperature. The solid product was filteredand washed with water (20 mL) and hexanes (10 mL), and dried under highvacuum.

General Procedure A-3:

A 15 mL pressure tube was charged with the aryl fluoride (1 mmol, 1 eq),water (5 mL), the nucleophile (1.1 eq) and sodium bicarbonate (2.1 eq).The tube was sealed and the reaction mixture was stirred at 90° C. for 2h. A precipitate appeared and the suspension was stirred overnight atroom temperature. The solid product was filtered and washed with water(20 mL) and hexanes (10 mL), and dried under high vacuum.

General Procedure A-4:

A 15 mL pressure tube was charged with the aryl fluoride (2.0 mmol, 1eq), dry dimethylformamide (5 mL), the nucleophile (4.0 mmol, 2 eq) andpotassium carbonate (829 mg, 6.0 mmol, 3 eq). The tube was sealed andthe reaction mixture was stirred vigorously at 90° C. for 10 h. At roomtemperature, the reaction mixture was diluted with dichloromethane (30mL) and sodium hydroxide 1 M (20 mL). The organic phase was washed with15% aq. lithium chloride (5×20 mL), dried over anhydrous magnesiumsulfate and concentrated under reduced pressure.

General Procedure B-1:

In a 10 mL oven-dried flask, the carboxylic acid DS-1-021 (1 mmol, 1 eq)was suspended in dry dichloromethane (5 mL). N-methyl morpholine (1 eq)was added dropwise and the resulting yellow homogeneous solution wascooled to 0° C. Isobutyl chloroformate (1 eq) was introduced dropwise.After 1 h, the amine (1.1 eq) was added dropwise and the reactionmixture was warmed to room temperature. After 4 h, dichloromethane (5mL) and water (5 mL) were introduced and the organic phase was washedwith water (10 mL), saturated sodium carbonate (3×10 mL). A precipitateappeared and was dissolved with water and the organic phase was washedwith brine (10 mL). The organic phase was dried over magnesium sulfate,filtered and concentrated under reduced pressure. Purification by columnchromatography on silica gel (0-10% MeOH in DCM, MeOH containing 1%Et₃N) afforded the pure product.

General Procedure B-2:

A 10 mL oven-dried flask was charged with the acyl chloride DS-1-059(312 mg, 1.0 mmol, 1 eq), dry dichloromethane (1 mL) and4-dimethylaminopyridine (2.5 mol %) and cooled to 0° C. A solution ofthe amine (1 eq) in dry dichloromethane (1 mL), was added dropwise over10 min and the reaction mixture was slowly warmed to room temperature.After 16 h, the reaction mixture was diluted with dichloromethane (10mL), washed with saturated sodium bicarbonate (2×5 mL), dried overmagnesium sulfate and concentrated under reduced pressure. Purificationby column chromatography on silica gel (0-20% MeOH in DCM, MeOHcontaining 1% NH₃) afforded the pure product.

General Procedure B-3:

A 50 mL oven-dried flask was charged with the carboxylic acid (30.0mmol, 1 eq) and thionyl chloride (7.5 mL) and heated to reflux. After 4h, the resulting homogeneous mixture was concentrated to dryness underreduced pressure. The residue obtained was then dissolved in dry tolueneor dichloromethane (5 mL) and the solvent was evaporated. This processwas repeated twice yielding the acyl chloride. Dry dichloromethane (20mL) and 4-dimethylaminopyridine (0.1 mol %) were introduced and theflask was cooled to 0° C. A solution of the amine (1 eq) in drydichloromethane (10 mL) was added dropwise over 15 min and the resultingsolution was slowly warmed to room temperature. After 3 h, a suspensionwas obtained and the solid was filtered, washed with diethyl ether (3×20mL) and dried under high vacuum to yield the pure product.

General Procedure C:

A 25 mL three-necked flask fitted with a reflux condenser was chargedwith DS-1-175 (492 mg, 1.3 mmol, 1 eq), the boronic acid (308 mg, 2.2mmol, 1.76 eq), potassium carbonate (829 mg, 6.0 mmol, 4.8 eq), water (2mL) and toluene (2.9 mL). The resulting suspension was degassed by 3freeze-pump-thaw cycles and the tetrakis(triphenylphosphine)palladium(0)(46 mg, 0.04 mmol, 3.2 mol %) was introduced. The reaction mixture washeated to reflux under argon and the homogeneous yellow solution wasvigorously stirred overnight. At room temperature, the reaction mixturewas filtered through Celite, and was washed with diethyl ether (25 mL).The organic phase was extracted with 2 M HCl (2×10 mL), the aqueouslayers were basified with solid potassium hydroxide to pH˜12-13 and wereextracted with dichloromethane (3×10 mL). The organic phases were driedover magnesium sulfate, filtered and concentrated under reducedpressure. Purification by column chromatography on silica gel (0-20%MeOH in DCM, MeOH containing 1% NH₃) afforded the pure product.

General Procedure D:

A 4 mL vial was charged with the amine and methanol (0.12 M). Hydrogenchloride was bubbled through the suspension until the solid entirelydissolved. The resulting homogeneous solution was concentrated underreduced pressure to afford the hydrochloride salt.

4-((4-fluorophenyl)thio)-3-nitrobenzoic acid (DS-1-021)

Following the general procedure A-1 using 4-fluoro-3-nitrobenzoic acid(10.0 mmol). Bright yellow microcrystalline powder (96%). ¹H NMR (400MHz, DMSO-d₆) δ 13.54 (bs, 1H), 8.61 (t, J=1.8 Hz, 1H), 8.00 (dt, J=8.5,1.8 Hz, 1H), 7.73-7.67 (m, 2H), 7.42 (td, J=8.8, 1.5 Hz, 2H), 6.90 (dd,J=8.6, 1.6 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.3, 163.6 (d,J_(CF)=249.3 Hz), 143.9, 143.4 (d, J_(CF)=1.0 Hz), 138.3 (d, J_(CF)=8.9Hz), 134.1, 128.2, 128.1, 126.5, 125.1 (d, J_(CF)=3.3 Hz), 117.8 (d,J_(CF)=22.1 Hz). IR (thin film): 3100, 1691, 1606, 1524, 1490, 1412,1337, 1239, 1159 cm⁻¹. MS (ES-API) m/z: 292.0 (100%, [M−H]⁻, C₁₃H₇FNO₄Srequires 292.0). mp: 214° C. (dec.).

4-((4-fluorophenyl)thio)-3-nitro-N-(pyridin-2-ylmethyl)benzamide(DS-1-023)

Following the general procedure B-1 using 2-picolylamine. Yellow solid(85%). ¹H NMR (400 MHz, DMSO-d₆) δ 9.42 (t, J=6.0 Hz, 1H), 8.83-8.78 (m,1H), 8.53-8.48 (m, 1H), 8.05 (dt, J=8.5, 1.4 Hz, 1H), 7.78-7.70 (m, 3H),7.44 (t, J=8.7 Hz, 2H), 7.32 (d, J=7.8 Hz, 1H), 7.27 (dd, J=7.5, 4.9 Hz,1H), 6.93 (d, J=8.5 Hz, 1H), 4.57 (d, J=5.8 Hz, 2H). ¹³C NMR (100 MHz,DMSO-d₆) δ 163.8, 163.5 (d, J_(CF)=249.1 Hz), 158.3, 148.9, 144.1, 141.5(d, J_(CF)=1.5 Hz), 138.2 (d, J_(CF)=8.9 Hz), 136.7, 132.7, 131.5,128.0, 125.4 (d, J_(CF)=3.2 Hz), 124.7, 122.2, 121.1, 117.8 (d,J_(CF)=22.1 Hz), 44.8. IR (thin film): 3072, 1651, 1645, 1607, 1590,1520, 1489, 1338, 1226, 1158 cm⁻¹. MS (ES-API) m/z: 384.1 (100%, [M+H]⁺,C₁₉H₁₅FN₃O₃S requires 384.1). mp: 140-141° C.

(R)—N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-1-031)

Following the general procedure B-1 using(R)-(+)-2-aminomethyl-1-ethylpyrrolidine. Yellow solid (46%). ¹H NMR(400 MHz, CDCl₃) δ 8.63 (t, J=2.4 Hz, 1H), 7.76 (dd, J=9.0, 2.6 Hz, 1H),7.59 (ddd, J=8.0, 5.1, 2.7 Hz, 2H), 7.21 (t, J=8.6 Hz, 2H), 6.87 (d,J=8.5 Hz, 2H), 3.71-3.63 (m, 1H), 3.34-3.26 (m, 1H), 3.19 (t, J=8.2 Hz,1H), 2.80 (td, J=7.9, 3.8 Hz, 1H), 2.68 (s, 1H), 2.31-2.15 (m, 2H),1.97-1.86 (m, 1H), 1.78-1.55 (m, 3H), 1.11 (t, J=7.4 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 164.9, 164.2 (d, J_(CF)=252.4 Hz), 144.4, 143.2,138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.6, 128.2, 125.6 (d, J_(CF)=3.6 Hz),124.5 (d, J_(CF)=2.5 Hz), 117.8 (d, J_(CF)=21.9 Hz), 62.1, 53.6, 48.1,40.9, 28.3, 23.1, 14.2. IR (thin film): 3097, 2971, 2877, 2807, 1652,1645, 1607, 1590, 1520, 1490, 1338, 1294, 1235, 1157 cm⁻¹. [α]_(D):+37.51° (0.885, MeOH). mp: 113° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-1-033)

Following the general procedure B-1 using2-aminomethyl-1-ethylpyrrolidine. Yellow solid (82%). ¹H NMR (400 MHz,CDCl₃) δ 8.62 (d, J=2.0 Hz, 1H), 7.75 (dd, J=8.5, 2.0 Hz, 1H), 7.61-7.55(m, 2H), 7.21 (t, J=8.5 Hz, 2H), 6.87 (d, J=8.5 Hz, 2H), 3.67 (ddd,J=13.8, 7.3, 2.6 Hz, 1H), 3.30 (ddd, J=13.7, 4.4, 2.5 Hz, 1H), 3.19 (td,J=8.3, 2.5 Hz, 1H), 2.80 (dq, J=12.0, 7.4 Hz, 1H), 2.72-2.64 (m, 1H),2.30-2.15 (m, 2H), 1.96-1.86 (m, 1H), 1.78-1.54 (m, 3H), 1.11 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 164.2 (d, J_(CF)=252.3 Hz),144.4, 143.2 (d, J_(CF)=1.6 Hz), 138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.6,128.2, 125.6 (d, J_(CF)=3.6 Hz), 124.5 (d, J_(CF)=1.7 Hz), 117.8 (d,J_(CF)=22.0 Hz), 62.0, 53.6, 48.1, 40.9, 28.4, 23.1, 14.2.

N-(2-(diethylamino)ethyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-1-043)

Following the general procedure B-2 using N,N-diethylethylenediamine.Yellow solid (32%). ¹H NMR (400 MHz, CDCl₃) δ 8.60 (t, J=1.9 Hz, 1H),7.74 (d, J=8.5 Hz, 1H), 7.61-7.52 (m, 2H), 7.23-7.15 (m, 2H), 7.07 (s,1H), 6.84 (dd, J=8.6, 1.8 Hz, 1H), 3.45 (q, J=4.8 Hz, 2H), 2.63 (t,J=5.4 Hz, 2H), 2.54 (q, J=7.1 Hz, 4H), 1.01 (t, J=7.0 Hz, 6H). ¹³C NMR(100 MHz, CDCl₃) δ 164.4, 164.2 (d, J_(CF)=252.3 Hz), 144.4, 143.2 (d,J_(CF)=1.6 Hz), 138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.6, 128.2 (d,J_(CF)=2.1 Hz), 125.6 (d, J_(CF)=3.6 Hz), 124.3 (d, J_(CF)=2.1 Hz),117.8 (d, J_(CF)=22.0 Hz), 51.0, 46.8, 37.5, 12.1. IR (thin film): 3096,2972, 2935, 2821, 1652, 1645, 1608, 1558, 1538, 1506, 1489, 1338, 1294,1227, 1158 cm⁻¹. MS (ES-API) m/z: 392.1 (100%, [M+H]⁺, C₁₉H₂₃FN₃O₃Srequires 392.1). mp: 98° C.

(S)—N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-1-053)

Following the general procedure B-2 using(S)-(−)-2-aminomethyl-1-ethylpyrrolidine. Yellow solid (82%). ¹H NMR(400 MHz, CDCl₃) δ 8.61 (d, J=2.0 Hz, 1H), 7.74 (dd, J=8.6, 2.0 Hz, 1H),7.60-7.53 (m, 2H), 7.22-7.16 (m, 2H), 7.03 (d, J=5.2 Hz, 1H), 6.84 (d,J=8.5 Hz, 1H), 3.65 (ddd, J=13.6, 7.3, 2.7 Hz, 1H), 3.27 (ddd, J=13.7,4.7, 2.8 Hz, 1H), 3.16 (ddd, J=9.5, 7.1, 2.6 Hz, 1H), 2.79 (dq, J=12.0,7.4 Hz, 1H), 2.66 (dddd, J=8.8, 6.9, 4.6, 2.7 Hz, 1H), 2.29-2.12 (m,2H), 1.89 (dtd, J=12.3, 8.5, 7.0 Hz, 1H), 1.77-1.52 (m, 3H), 1.09 (t,J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 164.2 (d, J_(CF)=252.4Hz), 144.4, 143.2, 138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.6, 128.2 (d,J_(CF)=1.5 Hz), 125.6, 124.5 (d, J_(CF)=2.0 Hz), 117.8 (d, J_(CF)=22.0Hz), 62.0, 53.6, 48.1, 40.9, 28.4, 23.1, 14.2. [α]_(D):—40.35° (1.13,MeOH).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(phenylthio)benzamide(DS-1-055)

Following the general procedure A-2 using thiophenol. Light yellow solid(74%). ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H), 7.74 (d, J=8.6 Hz, 1H),7.61-7.47 (m, 4H), 7.07-6.96 (m, 1H), 6.90 (d, J=8.5 Hz, 1H), 3.68 (dd,J=13.7, 7.6 Hz, 1H), 3.34-3.23 (m, 1H), 3.17 (t, J=8.0 Hz, 1H), 2.81(dq, J=15.0, 7.4 Hz, 1H), 2.71-2.62 (m, 1H), 2.22 (m, 2H), 2.03 (bs,1H), 1.89 (dt, J=16.9, 8.4 Hz, 1H), 1.79-1.53 (m, 3H), 1.11 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 144.5, 143.4, 136.1, 131.7,131.5, 130.6, 130.5, 130.4, 128.5, 124.4, 62.3, 53.6, 48.2, 40.8, 28.2,23.1, 14.2. IR (thin film): 2969, 2807, 1639, 1608, 1546, 1521, 1460,1338, 1294, 1241 cm⁻¹. MS (ES-API) m/z: 386.2 (100%, [M+H]⁺, C₂₀H₂₄N₃O₃Srequires 386.2). mp: 76° C.

4-((4-fluorophenyl)thio)-3-nitrobenzoyl chloride (DS-1-059)

A 25 mL oven-dried flask equipped with a reflux condenser was chargedwith DS-1-021 (1.47 g, 5.0 mmol, 1 eq) and thionyl chloride (10 mL) andheated to reflux overnight. The resulting homogeneous mixture was thenconcentrated to dryness under reduced pressure. The yellow solidobtained was then dissolved in dry toluene (5 mL) and the solvent wasevaporated. This process was repeated twice yielding the product (1.50g, 4.8 mmol, 96%) as a bright yellow microcrystalline powder. ¹H NMR(400 MHz, CDCl₃) δ 8.96 (t, J=1.6 Hz, 1H), 7.99 (d, J=8.7 Hz, 1H),7.63-7.56 (m, 2H), 7.25 (t, J=8.0 Hz, 2H), 6.94 (dd, J=8.8, 1.3 Hz, 1H).¹³C NMR (100 MHz, CDCl₃) δ 166.2, 164.5 (d, J_(CF)=253.3 Hz), 148.8 (d,J_(CF)=1.8 Hz), 144.4, 138.3 (d, J_(CF)=8.7 Hz), 134.2, 130.1, 129.0,128.4, 124.7 (d, J_(CF)=3.6 Hz), 118.2 (d, J_(CF)=22.1 Hz). IR (thinfilm): 1739, 1597, 1525, 1490, 1394, 1298, 1196, 1159, 970, 836, 731cm⁻¹. mp: 140° C.

4-(4-fluorophenyl)thio)-N-(2-(1-methylpyrrolidin-2-yl)ethyl)-3-nitrobenzamide(DS-1-061)

Following the general procedure B-2 using2-(2-aminoethyl)-1-methylpyrrolidine. Light yellow solid (84%). ¹H NMR(400 MHz, CDCl₃) δ 9.51 (s, 1H), 8.54 (d, J=1.2 Hz, 1H), 7.87 (dd,J=8.6, 1.2 Hz, 1H), 7.57 (dd, J=8.2, 5.4 Hz, 2H), 7.20 (t, J=8.4 Hz,2H), 6.84 (d, J=8.5 Hz, 1H), 3.74 (dq, J=15.1, 5.1 Hz, 1H), 3.49-3.38(m, 1H), 3.25-3.17 (m, 1H), 2.63-2.54 (m, 1H), 2.43 (s, 3H), 2.29 (q,J=7.6 Hz, 1H), 2.04-1.85 (m, 2H), 1.82-1.67 (m, 4H). ¹³C NMR (100 MHz,CDCl₃) δ 164.18 (d, J_(CF)=252.2 Hz), 163.9, 144.2, 143.0 (d, J_(CF)=1.6Hz), 138.3 (d, J_(CF)=8.7 Hz), 132.3, 131.9, 128.1, 125.6 (d, J_(CF)=3.6Hz), 123.8, 117.8 (d, J_(CF)=22.0 Hz), 65.5, 57.0, 40.5, 37.3, 27.6,27.4, 22.8. IR (thin film): 3072, 2945, 2877, 2789, 1652, 1645, 1608,1591, 1549, 1538, 1520, 1490, 1463, 1338, 1312, 1227, 1158 cm⁻¹. MS(ES-API) m/z: 404.1 (100%, [M+H]⁺, C₂₀H₂₃FN₃O₃S requires 404.1). mp:152-153° C. (dec.).

4-(4-fluorophenyl)thio)-3-nitro-N-(2-(pyrrolidin-1-yl)ethyl)benzamide(DS-1-063)

Following the general procedure B-2 using 1-(2-aminoethyl)pyrrolidine.Yellow solid (73%). ¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J=1.9 Hz, 1H),7.79 (dd, J=8.5, 1.9 Hz, 1H), 7.56 (m, 2H), 7.30 (s, 1H), 7.19 (t, J=8.6Hz, 2H), 6.83 (d, J=8.5 Hz, 1H), 3.56 (q, J=5.5 Hz, 2H), 2.77 (t, J=5.9Hz, 2H), 2.66-2.61 (m, 4H), 1.85-1.76 (m, 4H). ¹³C NMR (100 MHz, CDCl₃)δ 164.8, 164.2 (d, J_(CF)=252.3 Hz), 144.3, 143.2 (d, J_(CF)=1.6 Hz),138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.7, 128.1, 125.6 (d, J_(CF)=3.6 Hz),124.6, 117.8 (d, J_(CF)=22.0 Hz), 54.6, 54.0, 38.5, 23.6. IR (thinfilm): 2971, 2813, 1654, 1648, 1610, 1591, 1520, 1491, 1338, 1294, 1227,1158 cm⁻¹. MS (ES-API) m/z: 390.1 (100%, [M+H]⁺, C₁₉H₂₁FN₃O₃S requires390.1). mp: 173-175° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-((4-(trifluoromethyl)phenyl)thio)benzamide(DS-1-065)

Following the general procedure A-2 using 4-(trifluoromethyl)thiophenol.Light yellow solid (54%). ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 7.80(d, J=8.5 Hz, 1H), 7.72 (q, J=8.4 Hz, 4H), 7.24 (s, 1H), 6.90 (d, J=8.5Hz, 1H), 3.70 (ddd, J=13.7, 7.3, 2.3 Hz, 1H), 3.28 (dt, J=14.0, 3.4 Hz,1H), 3.15 (t, J=7.8 Hz, 1H), 2.87 (s, 1H), 2.81 (dq, J=14.7, 7.6 Hz,1H), 2.72-2.63 (m, 1H), 2.29-2.12 (m, 2H), 1.88 (dt, J=16.4, 8.1 Hz,1H), 1.77-1.52 (m, 2H), 1.09 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 164.7, 144.9, 141.1, 135.8, 135.4 (d, J_(CF)=1.6 Hz), 132.3, 132.2 (q,J_(CF)=33.0 Hz), 131.6, 128.6, 127.0 (q, J_(CF)=3.7 Hz), 124.5, 123.6(q, J_(CF)=272.6 Hz), 62.4, 53.5, 48.1, 40.8, 27.9, 22.9, 13.9. IR (thinfilm): 3091, 2972, 2877, 2803, 1634, 1607, 1557, 1520, 1465, 1398, 1326,1293, 1170, 1127, 1104, 1063, 846 cm⁻¹. MS (ES-API) m/z: 454.1 (100%,[M+H]⁺, C₂₁H₂₃F₃N₃O₃S requires 454.1). mp: 153-154° C. (dec.).

4-((3,4-difluorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-067)

Following the general procedure A-2 using 3,4-difluorothiophenol. Yellowsolid (90%). ¹H NMR (400 MHz, CDCl₃) δ 8.63 (s, 1H), 7.78 (d, J=8.4 Hz,1H), 7.43 (t, J=8.3 Hz, 1H), 7.40-7.26 (m, 2H), 7.01 (s, 1H), 6.89 (d,J=8.5 Hz, 1H), 3.67 (ddd, J=13.7, 7.3, 2.6 Hz, 1H), 3.29 (d, J=13.7 Hz,1H), 3.18 (t, J=7.2 Hz, 1H), 2.81 (dq, J=14.5, 7.2 Hz, 1H), 2.68 (s,1H), 2.32-2.13 (m, 2H), 1.98-1.85 (m, 1H), 1.79-1.53 (m, 3H), 1.11 (t,J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 152.1 (dd,J_(CF)=250.4, 8.1 Hz), 151.0 (dd, J_(CF)=253.2, 11.7 Hz), 144.5, 142.1,132.9 (dd, J_(CF)=6.6, 3.7 Hz), 132.3, 131.7, 128.2, 126.7 (t,J_(CF)=4.9 Hz), 125.0 (d, J_(CF)=17.4 Hz), 124.5, 119.3 (d, J_(CF)=17.6Hz), 62.0, 53.6, 48.1, 41.0, 28.4, 23.0, 14.2. IR (thin film): 3076,2972, 2878, 2807, 1652, 1645, 1607, 1520, 1505, 1464, 1407, 1338, 1276,1243, 1204, 1116, 1049 cm⁻¹. MS (ES-API) m/z: 422.1 (100%, [M+H]⁺,C₂₀H₂₂F₂N₃O₃S requires 422.1). mp: 122° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((3-fluorophenyl)thio)-3-nitrobenzamide(DS-1-069)

Following the general procedure A-2 using 3-fluorothiophenol. Yellowsolid (76%). ¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J=2.0 Hz, 1H), 7.77 (dd,J=8.5, 2.0 Hz, 1H), 7.49 (td, J=8.1, 5.7 Hz, 1H), 7.39 (dt, J=7.7, 1.3Hz, 1H), 7.32 (ddd, J=8.4, 2.5, 1.6 Hz, 1H), 7.23 (ddd, J=8.4, 2.6, 1.0Hz, 1H), 6.94 (d, J=8.5 Hz, 1H), 6.89 (s, 1H), 3.67 (ddd, J=13.7, 7.3,2.6 Hz, 1H), 3.30 (ddd, J=13.7, 4.5, 2.5 Hz, 1H), 3.19 (ddd, J=9.1, 7.1,2.3 Hz, 1H), 2.81 (dq, J=12.0, 7.4 Hz, 1H), 2.72-2.65 (m, 1H), 2.30-2.15(m, 2H), 1.92 (dq, J=12.4, 8.4 Hz, 1H), 1.80-1.53 (m, 3H), 1.11 (t,J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 163.3 (d, J_(CF)=251.3Hz), 144.7, 142.2, 132.5 (d, J_(CF)=7.3 Hz), 132.2, 131.8 (d, J_(CF)=8.2Hz), 131.7 (d, J_(CF)=3.1 Hz), 131.6, 128.5, 124.5, 122.7 (d,J_(CF)=21.8 Hz), 117.8 (d, J_(CF)=20.9 Hz), 62.0, 53.6, 48.1, 40.9,28.4, 23.1, 14.2. IR (thin film): 2971, 2810, 1645, 1609, 1521, 1472,1338, 1296, 1219, 1106 cm⁻¹. MS (ES-API) m/z: 404.1 (100%, [M+H]⁺,C₂₀H₂₃FN₃O₃S requires 404.1). mp: 103° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(pyridin-2-ylthio)benzamide(DS-1-071)

A 15 mL pressure tube was charged with the aryl fluoride DS-1-095 (164mg, 0.50 mmol, 1 eq), water (5 mL), 2-mercaptopyridine (61 mg, 0.55mmol, 1.1 eq) and sodium bicarbonate (47 mg, 0.55 mmol, 1.1 eq). Thetube was sealed and the reaction mixture was stirred at 90° C. for 2 hand then at room temperature overnight. The reaction mixture was dilutedwith ethyl acetate (8 mL) and water (2 mL) before sodium bicarbonate (10eq) was introduced. The organic phase was washed with water (2×4 mL) andbrine (4 mL), dried over magnesium sulfate and concentrated underreduced pressure to afford a brown oil. Purification by columnchromatography on silica gel (0-10% MeOH in DCM, MeOH containing 1% NH₃)afforded the product (87 mg, 0.22 mmol, 45%) as an orange oil. ¹H NMR(400 MHz, CDCl₃) δ 8.65-8.62 (m, 1H), 8.59 (s, 1H), 7.90-7.82 (m, 1H),7.80-7.70 (m, 1H), 7.58 (dd, J=7.9, 1.3 Hz, 1H), 7.37 (s, 1H), 7.34-7.30(m, 1H), 7.28-7.24 (m, 1H), 3.69 (ddd, J=13.9, 7.1, 3.6 Hz, 1H), 3.37(dt, J=13.9, 3.3 Hz, 1H), 3.26 (td, J=7.8, 3.0 Hz, 1H), 2.90-2.78 (m,2H), 2.39-2.23 (m, 2H), 2.00-1.89 (m, 1H), 1.81-1.68 (m, 2H), 1.69-1.55(m, 1H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.0,154.6, 151.3, 146.7, 138.4, 138.1, 133.0, 131.4, 130.9, 128.9, 124.4,123.7, 62.8, 53.7, 48.7, 41.0, 28.3, 23.1, 13.7. IR (thin film): 3051,2969, 2876, 2804, 1661, 1652, 1645, 1608, 1574, 1557, 1538, 1520, 1470,1455, 1421, 1338, 1311, 1246, 1150, 1108, 1049 cm⁻¹. MS (ES-API) m/z:387.1 (100%, [M+H]⁺, C₁₉H₂₃N₄O₃S requires 387.1).

N-(4-chloro-3-nitrobenzyl)-1-(1-ethylpyrrolidin-2-yl)methanamine(DS-1-073)

A 10 mL oven-dried flask was charged with 4-chloro-3-nitrobenzaldehyde(371 mg, 2.0 mmol, 1 eq), 2-(aminomethyl)-1-ethylpyrrolidine (0.29 mL,2.0 mmol, 1 eq), dry dichloromethane (4 mL) and acetic acid (0.23 mL,4.0 mmol, 2 eq). After stirring for 1 h at room temperature, sodiumtriacetoxyborohydride (636 mg, 3.0 mmol, 1.5 eq) was introduced in oneportion at 0° C. The reaction mixture was slowly warmed to roomtemperature and stirred overnight, upon which time it clogged. Saturatedsodium bicarbonate (5 mL) was carefully added followed bydichloromethane (6 mL). The organic phase was washed with saturatedsodium bicarbonate (5 mL), water (5 mL), dried over magnesium sulfateand concentrated under reduced pressure to afford a brown oil.Purification by column chromatography on silica gel (0-20% MeOH in DCM,MeOH containing 1% NH₃) yielded the product (170 mg, 0.58 mmol, 29%) asa light brown oil that was used in the next step without furtherpurification. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (s, 1H), 7.48 (dd, J=13.3,8.5 Hz, 2H), 3.84 (dd, J=18.6, 14.6 Hz, 2H), 3.18-3.09 (m, 1H), 2.78(dq, J=14.8, 7.4 Hz, 2H), 2.66-2.53 (m, 1H), 2.54-2.43 (m, 1H), 2.20(dq, J=11.3, 5.8 Hz, 1H), 2.13 (q, J=7.3 Hz, 1H), 1.93-1.83 (m, 1H),1.80-1.64 (m, 3H), 1.08 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ147.0, 140.9, 131.7, 130.7, 124.1, 123.9, 63.0, 53.0, 51.9, 51.4, 48.1,28.2, 22.1, 13.1. IR (thin film): 2969, 2874, 2799, 1569, 1538, 1479,1455, 1354, 1289, 1198, 1132, 1049 cm⁻¹.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((2-fluorophenyl)thio)-3-nitrobenzamide(DS-1-075)

Following the general procedure A-2 using 2-fluorothiophenol. Yellowsolid (88%). ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J=2.0 Hz, 1H), 7.78 (dd,J=8.5, 2.0 Hz, 1H), 7.64 (td, J=7.3, 1.8 Hz, 1H), 7.57 (dddd, J=8.2,7.1, 5.1, 1.8 Hz, 1H), 7.30 (td, J=7.6, 1.3 Hz, 1H), 7.26 (td, J=8.4,1.1 Hz, 1H), 6.90 (dd, J=8.5, 1.3 Hz, 2H), 3.67 (ddd, J=13.7, 7.3, 2.6Hz, 1H), 3.30 (ddd, J=13.8, 4.5, 2.6 Hz, 1H), 3.19 (ddd, J=9.5, 7.1, 2.5Hz, 1H), 2.81 (dq, J=12.0, 7.4 Hz, 1H), 2.68 (s, 1H), 2.30-2.15 (m, 2H),1.91 (dq, J=12.3, 8.3 Hz, 1H), 1.80-1.63 (m, 2H), 1.63-1.54 (m, 1H),1.11 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 163.2 (d,J_(CF)=251.0 Hz), 144.7, 141.3, 137.9, 133.4 (d, J_(CF)=8.1 Hz), 132.2,131.8, 128.0, 125.9 (d, J_(CF)=4.0 Hz), 124.6, 117.6 (d, J_(CF)=18.8Hz), 117.1 (d, J_(CF)=22.5 Hz), 62.0, 53.6, 48.1, 40.9, 28.3, 23.1,14.2. IR (thin film): 3076, 2970, 2876, 2805, 1652, 1645, 1608, 1549,1520, 1474, 1338, 1295, 1262, 1048 cm⁻¹. MS (ES-API) m/z: 404.1 (100%,[M+H]⁺, C₂₀H₂₃FN₃O₃S requires 404.1). mp: 136° C.

4-(4-acetamidophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-077)

Following the general procedure A-2 using 4-acetamidothiophenol (1.73mmol, 1.1 eq). Yellow-orange solid (93%). ¹H NMR (400 MHz, CDCl₃) δ 8.62(d, J=2.0 Hz, 1H), 7.72 (dd, J=8.5, 2.0 Hz, 1H), 7.67 (d, J=8.5 Hz, 2H),7.55-7.51 (m, 2H), 7.45-7.39 (m, 1H), 6.93 (s, 1H), 6.90 (d, J=8.5 Hz,1H), 3.67 (ddd, J=13.7, 7.3, 2.6 Hz, 1H), 3.30 (ddd, J=13.7, 4.5, 2.6Hz, 1H), 3.20 (ddd, J=9.2, 6.8, 2.4 Hz, 1H), 2.81 (dq, J=12.0, 7.4 Hz,1H), 2.72-2.65 (m, 1H), 2.24 (s, 3H), 2.30-2.16 (m, 1H), 1.91 (dq,J=12.4, 8.4 Hz, 1H), 1.79-1.53 (m, 4H), 1.11 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 168.9, 165.3, 144.5, 144.2, 140.4, 137.1, 131.3,130.9, 128.4, 125.2, 124.4, 121.2, 64.6, 53.9, 49.8, 40.7, 28.1, 24.9,23.4, 12.7. IR (thin film): 3101, 2969, 2808, 1646, 1607, 1591, 1520,1464, 1397, 1371, 1338, 1312, 1292, 1257, 1179 cm⁻¹. MS (ES-API) m/z:443.2 (100%, [M+H]⁺, C₂₂H₂₇N₄O₄S requires 443.2). mp: 138-140° C.(dec.).

1-(1-ethylpyrrolidin-2-yl)-N-(4-((4-fluorophenyl)thio)-3-nitrobenzyl)methanamine(DS-1-079)

A 15 mL pressure tube was charged with the aryl chloride DS-1-073 (170mg, 0.58 mmol, 1 eq), water (6 mL), 4-fluorothiophenol (0.07 mL, 0.64mmol, 1.1 eq) and sodium bicarbonate (54 mg, 0.64 mmol, 1.1 eq). Thetube was sealed and the reaction mixture was stirred at 90° C. for 2 hand then overnight at room temperature. The reaction mixture was dilutedwith saturated sodium bicarbonate (4 mL) and the aqueous phase wasextracted with hexane (3×5 mL) and dichloromethane (3×5 mL). Thecombined organic phase was washed with water (2×15 mL), dried overmagnesium sulfate and concentrated under reduced pressure to afford abrown oil. Purification by column chromatography on silica gel (0-10%MeOH in DCM, MeOH containing 1% NH₃) afforded the product (206 mg, 0.53mmol, 91%) as an orange-red oil. ¹H NMR (400 MHz, CDCl₃) δ 8.20 (d,J=1.9 Hz, 1H), 7.56 (ddd, J=8.7, 5.3, 0.9 Hz, 2H), 7.35 (dd, J=8.4, 0.8Hz 1H), 7.17 (t, J=8.4 Hz, 2H), 6.75 (d, J=8.4 Hz, 1H), 3.81 (s, 2H),3.17-3.10 (m, 1H), 2.83-2.72 (m, 1H), 2.63 (dd, J=11.2, 3.7 Hz, 1H),2.59-2.49 (m, 1H), 2.50-2.42 (m, 1H), 2.23-2.07 (m, 2H), 1.93-1.83 (m,1H), 1.78-1.62 (m, 3H), 1.57 (s, 1H), 1.07 (t, J=7.1 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) □ 163.8 (d, J_(CF)=251.3 Hz), 144.9, 139.1, 138.1 (d,J_(CF)=8.5 Hz), 137.2 (d, J_(CF)=1.5 Hz), 133.4, 128.1, 126.6 (d,J_(CF)=3.6 Hz), 125.0, 117.4 (d, J_(CF)=21.9 Hz), 64.0, 53.9, 52.8,52.4, 49.0, 29.1, 23.0, 14.0. IR (thin film): 2967, 2798, 1590, 1556,1519, 1490, 1336, 1291, 1226, 1156, 1108, 1048, 834 cm⁻¹. MS (ES-API)m/z: 390.2 (100%, [M+H]⁺, C₂₀H₂₅FN₃O₂S requires 390.2).

(S)-4-((4-fluorophenyl)thio)-3-nitro-N-(pyrrolidin-2-ylmethyl)benzamide(DS-1-085)

A 10 mL oven-dried flask was charged with(S)-(+)-2-(aminomethyl)pyrrolidine (0.16 mL, 1.50 mmol, 1.5 eq) and drydichloromethane (2.5 mL) and immersed in an ice bath. A solution of theacyl chloride DS-1-059 (312 mg, 1.00 mmol, 1 eq) in dry dichloromethane(2.5 mL) was then added dropwise. After 14 h, saturated sodiumbicarbonate (5 mL) was introduced and the organic phase was washed withsaturated sodium bicarbonate (2×5 mL) and water (5 mL), dried overmagnesium sulfate, filtered and concentrated to obtain a brown oil.Purification by column chromatography on silica gel (0-20% MeOH in DCM,MeOH containing 1% NH₃) afforded the product (27 mg, 0.07 mmol, 7%) as ayellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.40 (d, J=1.9 Hz, 1H), 7.61-7.50(m, 3H), 7.19 (t, J=8.5 Hz, 2H), 6.82 (d, J=8.4 Hz, 1H), 4.31-4.20 (m,1H), 3.55-3.40 (m, 2H), 3.07-3.01 (m, 1H), 2.86 (dd, J=12.5, 6.6 Hz,1H), 2.12 (dt, J=10.7, 6.4 Hz, 1H), 1.98-1.73 (m, 3H), 1.40 (s, 2H). ¹³CNMR (100 MHz, CDCl₃) δ 167.5, 164.2 (d, J_(CF)=252.3 Hz), 144.1, 141.9,138.3 (d, J_(CF)=8.8 Hz), 134.1, 132.5, 128.1, 125.7, 125.0, 117.8 (d,J_(CF)=22.0 Hz), 60.4, 50.8, 44.7, 28.5, 25.2. IR (thin film): 3069,2969, 2875, 1622, 1608, 1590, 1548, 1520, 1491, 1418, 1337, 1292, 1225,1157, 1049 cm⁻¹. MS (ES-API) m/z: 376.1 (100%, [M+H]⁺, C₁₈H₁₉FN₃O₃Srequires 376.1). [α]_(D): −89.02 (c 1.35, CHCl₃).

4-((4-fluorophenyl)thio)-N-(2-morpholinoethyl)-3-nitrobenzamide(DS-1-089)

Following the general procedure B-2 using 4-(2-aminoethyl)morpholine.Yellow solid (66%). ¹H NMR (400 MHz, CDCl₃) δ 8.61 (d, J=2.0 Hz, 1H),7.78 (dd, J=8.5, 2.0 Hz, 1H), 7.61-7.55 (m, 2H), 7.25-7.18 (m, 2H), 6.88(d, J=8.5 Hz, 1H), 6.78 (s, 1H), 3.75-3.70 (m, 4H), 3.55 (q, J=5.6 Hz,2H), 2.60 (t, J=6.0 Hz, 2H), 2.53-2.47 (m, 4H). ¹³C NMR (100 MHz, CDCl₃)δ 164.7, 164.2 (d, J_(CF)=252.4 Hz), 144.3, 143.5 (d, J_(CF)=1.6 Hz),138.3 (d, J_(CF)=8.7 Hz), 131.8, 131.6, 128.3, 125.5 (d, J_(CF)=3.6 Hz),124.3, 117.8 (d, J_(CF)=22.0 Hz), 67.1, 56.8, 53.4, 36.3. IR (thinfilm): 3095, 2955, 2856, 2814, 1643, 1608, 1590, 1548, 1520, 1491, 1338,1296, 1227, 1158, 1117, 837 cm⁻¹. MS (ES-API) m/z: 406.1 (100%, [M+H]⁺,C₁₉H₂₁FN₃O₄S requires 406.1). mp: 159-161° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-fluoro-3-nitrobenzamide (DS-1-095)

A 50 mL oven-dried flask equipped with a reflux condenser, fitted with adrying tube, was charged with 4-fluoro-3-nitrobenzoic acid (5.56 g, 30.0mmol, 1 eq) and thionyl chloride (7.5 mL) and heated to reflux. After 4h, the resulting homogeneous mixture was concentrated to dryness underreduced pressure. The colorless liquid obtained was then dissolved indry toluene (5 mL) and the solvent was evaporated. This process wasrepeated twice yielding 4-fluoro-3-nitrobenzoyl chloride (4.06 g, 19.9mmol, quantitative) as a colorless liquid.

Dry dichloromethane (30 mL) was introduced and the flask was immersed ina water bath. To this solution was added dropwise2-(aminomethyl)-1-ethylpyrrolidine (3.7 mL, 27.0 mmol, 0.9 eq). After 14h, the resulting beige suspension was diluted with dichloromethane (20mL) and saturated sodium bicarbonate (50 mL) and the aqueous layer wasbasified to pH˜9 with a concentrated potassium hydroxide solution. Theorganic phase was washed with saturated sodium bicarbonate (2×50 mL) andwater (50 mL), dried over magnesium sulfate, filtered and concentratedto obtain a brown oil. Purification by column chromatography on silicagel (0-20% MeOH in DCM, MeOH containing 1% NH₃) afforded the crudeproduct that was dissolved in dichloromethane (20 mL) and extracted withHCl 2 M (3×10 mL). The aqueous phases were washed with dichloromethane(20 mL), basified to pH˜13 with a concentrated potassium hydroxidesolution and extracted with dichloromethane (3×10 mL). The organiclayers were washed with sodium bicarbonate (20 mL), water (20 mL), driedover magnesium sulfate, filtered and concentrated to yield the pureproduct (6.00 g, 20.3 mmol, 75%) as a light brown oil which darkens overdays. ¹H NMR (400 MHz, CDCl₃) δ 8.45 (dd, J=7.0, 2.3 Hz, 1H), 8.05 (ddd,J=8.6, 4.2, 2.3 Hz, 1H), 7.39-7.28 (m, 1H), 7.20 (bs, 1H), 3.63 (ddd,J=13.6, 7.1, 2.7 Hz, 1H), 3.31-3.22 (m, 1H), 3.19-3.11 (m, 1H), 2.79(dq, J=14.7, 7.3 Hz, 1H), 2.71-2.61 (m, 1H), 2.27-2.13 (m, 2H), 1.88(ddd, J=16.4, 12.0, 8.3 Hz, 1H), 1.76-1.49 (m, 3H), 1.07 (t, J=7.2 Hz,3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 157.0 (d, J_(CF)=269.5 Hz), 137.1(d, J_(CF)=7.7 Hz), 134.2 (d, J_(CF)=9.4 Hz), 131.8, 125.1 (d,J_(CF)=2.1 Hz), 118.8 (d, J_(CF)=21.3 Hz), 62.1, 53.5, 48.1, 41.3, 28.3,22.9, 14.0. IR (thin film): 3090, 2970, 2877, 2802, 1646, 1620, 1538,1494, 1351, 1316, 1268 cm⁻¹. MS (ES-API) m/z: 296.2 (100%, [M+H]⁺,C₁₄H₁₉FN₃O₃ requires 296.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-(4-fluorophenoxy)-3-nitrobenzamide(DS-1-103)

Following the general procedure A-2 using 4-fluorophenol. Purificationby column chromatography on silica gel (0-20% MeOH in DCM, MeOHcontaining 1% NH₃). Beige solid (48%). ¹H NMR (400 MHz, CDCl₃) δ 8.36(d, J=2.2 Hz, 1H), 7.93 (dd, J=8.7, 2.2 Hz, 1H), 7.16 (bs, 1H),7.14-7.03 (m, 4H), 6.95 (d, J=8.7 Hz, 1H), 3.68 (ddd, J=13.7, 7.2, 3.0Hz, 1H), 3.33 (dt, J=13.7, 3.5 Hz, 1H), 3.26-3.19 (m, 1H), 2.89-2.79 (m,1H), 2.78-2.72 (m, 1H), 2.37-2.19 (m, 2H), 2.01-1.86 (m, 1H), 1.82-1.55(m, 3H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 160.1(d, J_(CF)=244.8 Hz), 153.5, 150.5 (d, J_(CF)=2.7 Hz), 140.3, 132.7,129.4, 124.9, 121.8 (d, J_(CF)=8.5 Hz), 118.8, 117.1 (d, J_(CF)=23.6Hz), 62.6, 53.7, 48.4, 40.9, 28.3, 23.1, 13.9. IR (thin film): 3078,2971, 2877, 2804, 1652, 1645, 1623, 1538, 1532, 1504, 1487, 1354, 1266,1226, 1188, 1148, 1090 cm⁻¹. MS (ES-API) m/z: 388.2 (100%, [M+H]⁺,C₂₀H₂₃FN₃O₄ requires 388.2). mp: 91-94° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)amino)-3-nitrobenzamide(DS-1-105)

Following the general procedure A-2 using 4-fluoroaniline. Purificationby column chromatography on silica gel (0-10% MeOH in DCM, MeOHcontaining 1% NH₃). Orange solid (71%). ¹H NMR (400 MHz, CDCl₃) δ 9.60(s, 1H), 8.68 (s, 1H), 7.87 (d, J=8.6 Hz, 1H), 7.32-7.22 (m, 2H),7.19-7.12 (m, 2H), 7.06 (d, J=9.0 Hz, 1H), 3.69 (ddd, J=13.6, 7.1, 3.4Hz, 1H), 3.42-3.25 (m, 2H), 2.93-2.77 (m, 2H), 2.43-2.24 (m, 2H), 1.95(dt, J=17.0, 8.5 Hz, 1H), 1.84-1.61 (m, 3H), 1.17 (t, J=7.1 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.5, 161.2 (d, J_(CF)=247.2 Hz), 145.5, 134.4,133.9 (d, J_(CF)=3.2 Hz), 132.1, 127.5 (d, J_(CF)=8.4 Hz), 125.9, 123.8,117.0 (d, J_(CF)=22.8 Hz), 115.8, 62.7, 53.7, 48.5, 40.9, 28.3, 23.2,14.0. IR (thin film): 3077, 2970, 2877, 2802, 1651, 1622, 1558, 1506,1411, 1353, 1268, 1212, 1153, 1072 cm⁻¹. MS (ES-API) m/z: 387.2 (100%,[M+H]⁺, C₂₀H₂₄FN₄O₃ requires 387.2). mp: 120° C.

3-((4-fluorophenyl)thio)-4-nitrobenzoic acid (DS-1-113)

Following the general procedure A-1 using 3-fluoro-4-nitrobenzoic acid(10.0 mmol). Yellow needles (100%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.64(s, 1H), 8.34 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.5 Hz, 1H), 7.74 (dd,J=8.3, 5.5 Hz, 2H), 7.45 (t, J=8.7 Hz, 2H), 7.39 (s, 1H). IR (thinfilm): 1698, 1574, 1510, 1423, 1334, 1288, 1088, 822 cm⁻¹. MS (ES-API)m/z: 291.9 (100%, [M−H]⁻, C₁₃H₇FNO₄S requires 292.0), 585.0 (12%,[2M−H]⁻), 607.0 (17%, [2M+Na−2H]⁻).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)benzamide(DS-1-117)

A 20 mL oven-dried Schlenk tube was charged with potassium tert-butoxide(269 mg, 2.40 mmol, 2.4 eq) and heated to 110° C. under high vacuum.After 30 min, 4-bromobenzoic acid was introduced at room temperature(201 mg, 1.00 mmol, 1 eq) and the solid blend was heated to 110° C.under high vacuum. After 30 min, dry toluene (6 mL) was added underargon at room temperature. A 4 mL oven-dried vial was charged withpalladium acetate (6.7 mg, 0.03 mmol, 3 mol %) and Josiphos CyPF-tBu(16.6 mg, 0.03 mmol, 3 mol %). The vial was then evacuated andback-filled with argon three times before introducing dry toluene (3mL). After 1 min, the resulting orange solution was added in one portionto the white suspension in the Schlenk tube, immediately followed by4-fluorothiophenol (0.13 mL, 1.20 mmol, 1.2 eq) in one portion. TheSchlenk tube was sealed with a screw cap and heated to 110° C. After 16h, the resulting brown suspension was cooled to room temperature, silicagel was introduced (500 mg) and the solvent was evaporated under reducedpressure. Purification by column chromatography on silica gel (0-20%EtOAc in hexanes, EtOAc containing 4% AcOH) afforded the product (203mg) in a 3:1 mixture with 4-bromobenzoic acid as a white solid.

A 10 mL oven-dried flask equipped with a reflux condenser was chargedwith the mixture of carboxylic acids (203 mg) obtained from the previousstep and thionyl chloride (3 mL) and heated to reflux overnight. Theresulting homogeneous mixture was then concentrated to dryness underreduced pressure. The colorless liquid obtained was then dissolved indry toluene (2.5 mL) and the solvent was evaporated. This process wasrepeated twice yielding the mixture of acyl chlorides as a colorlessoil.

Dry dichloromethane (4 mL) was introduced and2-(aminomethyl)-1-ethylpyrrolidine (0.10 mL, 0.72 mmol, 0.72 eq) wasadded dropwise. After 8 h, the solution was diluted with dichloromethane(6 mL) and saturated sodium bicarbonate (10 mL). The organic phase waswashed with saturated sodium bicarbonate (2×10 mL) and water (10 mL),dried over magnesium sulfate, filtered and concentrated to obtain abrown oil. Purification by column chromatography on silica gel (0-10%MeOH in DCM, MeOH containing 1% NH₃) afforded the desired amide (20 mg,0.056 mmol, 8% over 3 steps) as a light brown oil. ¹H NMR (400 MHz,CDCl₃) δ 7.70 (d, J=8.5 Hz, 2H), 7.49-7.43 (m, 2H), 7.22-7.13 (m, 2H),7.11-7.04 (m, 2H), 3.69 (ddd, J=13.8, 7.3, 3.6 Hz, 1H), 3.35 (dt,J=13.8, 3.5 Hz, 1H), 3.27 (dt, J=9.5, 4.7 Hz, 1H), 2.93-2.78 (m, 2H),2.41-2.22 (m, 2H), 1.93 (dq, J=12.2, 8.0 Hz, 1H), 1.81-1.58 (m, 3H),1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 167.1, 163.1 (d,J_(CF)=249.4 Hz), 142.3, 136.0 (d, J_(CF)=8.4 Hz), 132.1, 131.9, 128.8,127.8 (d, J_(CF)=4.3 Hz), 116.9 (d, J_(CF)=22.1 Hz), 63.1, 53.7, 48.7,40.7, 28.2, 23.2, 13.7. IR (thin film): 3063, 2969, 2875, 2801, 1652,1644, 1634, 1595, 1538, 1488, 1397, 1296, 1225, 1156, 1085, 1014, 834,758 cm⁻¹. MS (ES-API) m/z: 359.2 (100%, [M+H]⁺, C₂₀H₂₄FN₂OS requires359.2).

1-ethyl-2-((4-((4-fluorophenyl)thio)-3-nitrobenzamido)methyl)-1-methylpyrrolidin-1-iumiodide (DS-1-119)

A 5 mL flask was charged with DS-1-033 (171 mg, 0.42 mmol, 1 eq),ethanol (1 mL) and iodomethane (80 μL, 0.85 mmol, 2 eq). The yellowsolution was stirred for 3 h and the resulting suspension was filtered,washed with ethanol (1 mL) and diethyl ether (2 mL) to afford a yellowsolid. Purification by column chromatography on silica gel (0-50% MeOHin DCM) yielded the desired ammonium salt (39 mg, 0.07 mmol, 17%) as amixture of two diastereoisomers as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.97-8.86 (m, 1H), 8.85 (d, J=1.9 Hz, 1H), 8.17 (dt, J=8.6, 2.5Hz, 1H), 7.57-7.49 (m, 2H), 7.22-7.15 (m, 2H), 6.82 (d, J=8.6 Hz, 1H),4.54-4.45* (m), 4.43-4.33 (m, 1H), 4.12-3.75 (m, 3H), 3.73-3.49 (m, 3H),3.39-3.32* (m), 3.28* (s), 3.27 (s, 3H), 3.23-3.16* (m), 2.57-2.48 (m,1H), 2.25-2.08 (m, 3H), 1.45* (t, J=7.2 Hz), 1.41 (t, J=7.3 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.4, 164.2 (d, J_(CF)=266.8 Hz), 144.2, 143.9(d, J_(CF)=1.5 Hz), 138.2 (d, J_(CF)=8.6 Hz), 132.3, 129.7, 128.1,125.9, 125.1 (d, J_(CF)=3.5 Hz), 117.9 (d, J_(CF)=22.0 Hz), 75.8*, 73.6,64.5, 62.4*, 59.8, 51.3*, 49.0*, 46.5*, 43.5, 37.9, 37.6*, 26.2, 19.2*,18.9, 9.8, 8.7*. * minor diastereoisomer. IR (thin film): 3260, 2940,1652, 1607, 1590, 1520, 1489, 1465, 1397, 1338, 1308, 1225, 1157, 749cm⁻¹. MS (ES-API) m/z: 418.2 (100%, [M+H]⁺, C₂₁H₂₅FN₃O₃S requires418.2). mp: 95-97° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-isopropylphenyl)thio)-3-nitrobenzamide(DS-1-123)

Following the general procedure A-2 using 4-isopropylthiophenol.Purification by column chromatography on silica gel (0-20% MeOH in DCM,MeOH containing 1% NH₃). Yellow solid (28%). ¹H NMR (400 MHz, CDCl₃) δ8.66 (d, J=1.8 Hz, 1H), 7.78 (dd, J=8.6, 1.8 Hz, 1H), 7.47 (d, J=8.1 Hz,2H), 7.40 (bs, 1H), 7.34 (d, J=8.1 Hz, 2H), 6.91 (d, J=8.5 Hz, 1H), 3.67(ddd, J=13.8, 7.1, 3.7 Hz, 1H), 3.36 (dt, J=13.9, 3.4 Hz, 1H), 3.31-3.21(m, 1H), 2.97 (hept, J=7.0 Hz, 1H), 2.93-2.79 (m, 2H), 2.42-2.23 (m,2H), 1.94 (dq, J=12.5, 8.4 Hz, 1H), 1.81-1.58 (m, 3H), 1.29 (d, J=6.9Hz, 6H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1,151.7, 144.4, 144.0, 136.0, 131.29, 131.26, 128.6, 128.4, 126.8, 124.7,63.0, 53.6, 48.7, 40.9, 34.1, 28.3, 23.9, 23.1, 13.6. IR (thin film):2963, 1654, 1605, 1559, 1520, 1490, 1458, 1386, 1339, 1241, 1100, 1049cm⁻¹. MS (ES-API) m/z: 428.2 (100%, [M+H]⁺, C₂₃H₂₉N₃O₃S requires 428.2).mp: 93-94° C.

4-((2,4-difluorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-125)

Following the general procedure A-2 using 2,4-difluorothiophenol. Yellowsolid (77%). ¹H NMR (400 MHz, CDCl₃) δ 8.69 (s, 1H), 7.85 (d, J=8.5 Hz,1H), 7.63 (td, J=8.4, 6.3 Hz, 1H), 7.09-6.99 (m, 2H), 6.87 (dd, J=8.5,1.0 Hz, 1H), 3.70 (ddd, J=14.0, 7.4, 3.0 Hz, 1H), 3.41-3.32 (m, 1H),3.31-3.21 (m, 1H), 2.91-2.73 (m, 2H), 2.38-2.24 (m, 2H), 2.01-1.90 (m,1H), 1.82-1.59 (m, 3H), 1.15 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 165.2 (dd, J_(CF)=255.1, 11.3 Hz), 164.9, 163.8 (dd, J_(CF)=253.4,12.5 Hz), 144.7, 141.0, 139.0 (dd, J_(CF)=9.9, 1.7 Hz), 132.2, 132.0,127.6, 124.8, 113.5 (dd, J_(CF)=21.6, 3.9 Hz), 113.3 (dd, J_(CF)=19.1,4.1 Hz), 105.8 (dd, J_(CF)=26.1, 26.1 Hz), 62.5, 53.6, 48.4, 40.8, 28.1,23.1, 14.0. IR (thin film): 2969, 1648, 1608, 1546, 1522, 1485, 1466,1420, 1339, 1294, 1267, 1241, 1144, 967 cm⁻¹. MS (ES-API) m/z: 422.1(100%, [M+H]⁺, C₂₀H₂₁F₂N₃O₃S requires 422.1). mp: 63-65° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(pyridin-4-ylthio)benzamide(DS-1-129)

A 15 mL pressure tube was charged with DS-1-095 (295 mg, 1.00 mmol, 1eq), water (5 mL), 4-mercaptopyridine (145 mg, 1.30 mmol, 1.3 eq) andsodium bicarbonate (109 mg, 1.30 mmol, 1.3 eq). The tube was sealed andthe reaction mixture was stirred at 90° C. for 2 h and at roomtemperature overnight. The reaction mixture was diluted withdichloromethane (20 mL) and the organic layer was washed with saturatedsodium bicarbonate (2×20 mL) and then extracted with 2M HCl (3×10 mL).The acidic aqueous phases were basified to pH˜12 with a concentratedpotassium hydroxide solution and extracted with dichloromethane (3×10mL). The organic layers were dried over magnesium sulfate, filtered andsilica gel (1 g) was introduced before the solvent was evaporated underreduced pressure. Purification by column chromatography on silica gel(0-20% MeOH in DCM, MeOH containing 1% NH₃) afforded the product (191mg, 0.49 mmol, 49%) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ8.69-8.65 (m, 2H), 8.60 (d, J=1.9 Hz, 1H), 7.83 (dd, J=8.5, 2.0 Hz, 1H),7.44-7.39 (m, 2H), 7.13 (d, J=8.4 Hz, 1H), 7.13 (bs, 1H), 3.68 (ddd,J=13.8, 7.3, 2.8 Hz, 1H), 3.35-3.28 (m, 1H), 3.24-3.17 (m, 1H), 2.82(dq, J=12.1, 7.4 Hz, 1H), 2.76-2.70 (m, 1H), 2.32-2.19 (m, 2H), 1.92(dq, J=12.4, 8.3 Hz, 1H), 1.80-1.54 (m, 3H), 1.11 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 164.6, 151.2, 146.3, 142.4, 138.4, 133.4, 131.7,130.0, 128.2, 124.5, 62.3, 53.6, 48.3, 40.9, 28.3, 23.1, 14.0. IR (thinfilm): 2968, 2801, 1660, 1652, 1608, 1573, 1538, 1520, 1470, 1404, 1339,1294, 1213, 1048, 749 cm⁻¹. MS (ES-API) m/z: 387.1 (36%, [M+H]⁺,C₁₉H₂₃N₄O₃S requires 387.1). mp: 155-157° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorobenzyl)thio)-3-nitrobenzamide(DS-1-131)

Following the general procedure A-2 using 4-fluorobenzyl mercaptan.Yellow solid (70%). ¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J=1.9 Hz, 1H),7.96 (dd, J=8.4, 2.0 Hz, 1H), 7.49 (d, J=8.5 Hz, 1H), 7.42-7.35 (m, 2H),7.07-7.00 (m, 2H), 7.01 (bs, 1H), 4.20 (s, 2H), 3.68 (ddd, J=13.7, 7.2,2.7 Hz, 1H), 3.32 (ddd, J=13.7, 4.4, 2.9 Hz, 1H), 3.25-3.17 (m, 1H),2.83 (dq, J=12.1, 7.4 Hz, 1H), 2.74-2.67 (m, 1H), 2.33-2.17 (m, 2H),1.93 (dq, J=12.4, 8.3 Hz, 1H), 1.81-1.56 (m, 3H), 1.13 (t, J=7.2 Hz,3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 162.5 (d, J_(CF)=247.2 Hz),145.2, 141.5, 132.0, 131.5, 130.8 (d, J_(CF)=8.2 Hz), 130.2 (d,J_(CF)=3.3 Hz), 126.9, 124.5, 116.0 (d, J_(CF)=21.7 Hz), 62.2, 53.7,48.2, 41.0, 36.9, 28.4, 23.1, 14.2. IR (thin film): 3075, 2969, 2876,2801, 1652, 1607, 1548, 1510, 1465, 1338, 1292, 1230, 1158, 1107, 1052cm⁻¹. MS (ES-API) m/z: 418.2 (100%, [M+H]⁺, C₂₁H₂₅FN₃O₃S requires418.2). mp: 142-143° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-methoxyphenyl)thio)-3-nitrobenzamide(DS-1-133)

Following the general procedure A-2 using 4-methoxythiophenol. Yellowsolid (53%). ¹H NMR (400 MHz, CDCl₃) δ 8.65 (d, J=1.9 Hz, 1H), 7.76 (dd,J=8.6, 2.0 Hz, 1H), 7.52-7.46 (m, 2H), 7.08 (bs, 1H), 7.05-6.99 (m, 2H),6.89 (d, J=8.6 Hz, 1H), 3.88 (s, 3H), 3.69 (ddd, J=13.8, 7.3, 3.0 Hz,1H), 3.32 (dt, J=13.8, 3.5 Hz, 1H), 3.24-3.18 (m, 1H), 2.83 (dq, J=12.1,7.4 Hz, 1H), 2.78-2.68 (m, 1H), 2.32-2.19 (m, 2H), 1.92 (dq, J=12.3, 8.3Hz, 1H), 1.81-1.55 (m, 3H), 1.12 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 165.1, 161.6, 144.6, 144.3, 137.8, 131.5, 131.4, 128.3, 124.5,120.6, 116.0, 62.5, 55.6, 53.7, 48.4, 40.8, 28.2, 23.1, 14.0. IR (thinfilm): 2969, 2837, 1648, 1607, 1592, 1546, 1520, 1494, 1461, 1338, 1290,1251, 1173, 1106, 1048, 1028 cm⁻¹. MS (ES-API) m/z: 416.2 (100%, [M+H]⁺,C₂₁H₂₆N₃O₄S requires 416.2). mp: 69-71° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)sulfinyl)-3-nitrobenzamide(DS-1-135)

A 15 mL pressure tube was charged with DS-1-033 (101 mg, 0.25 mmol, 1eq), acetic acid (1.25 mL), and 35% aqueous hydrogen peroxide (31 mg,0.33 mmol, 1.3 eq). The tube was sealed and the homogeneous solution wasstirred at 60° C. After 16 h, the solution is cooled to roomtemperature, poured into ice-water (20 mL) and basified to pH˜11 with30% aqueous ammonium hydroxide. The resulting precipitate was filteredand washed with water (10 mL) to afford the product (57 mg, 0.14 mmol,54%) as a 1:1 mixture of diastereoisomers as a light brown solid. ¹H NMR(400 MHz, CDCl₃) δ 8.65 (dd, J=2.9, 1.7 Hz, 1H), 8.61 (d, J=8.2 Hz, 1H),8.38 (ddd, J=8.2, 2.7, 1.8 Hz, 1H), 7.73-7.67 (m, 2H), 7.34 (bs, 1H),7.13-7.03 (m, 2H), 3.70 (ddd, J=13.6, 7.1, 2.8 Hz, 1H), 3.34 (dt,J=13.8, 3.5 Hz, 1H), 3.26-3.17 (m, 1H), 2.88-2.79 (m, 1H), 2.79-2.72 (m,1H), 2.35-2.20 (m, 2H), 2.00-1.89 (m, 1H), 1.81-1.56 (m, 3H), 1.13 (t,J=7.2 Hz, 1.5H), 1.12* (t, J=7.2 Hz, 1.5H). ¹³C NMR (100 MHz, CDCl₃) δ164.5 (d, J_(CF)=253.7 Hz), 164.3, 146.6, 144.7, 140.4 (d, J_(CF)=3.3Hz), 138.6, 133.5 (d, J_(CF)=8.5 Hz), 129.4 (d, J_(CF)=9.0 Hz), 126.7,124.4 (d, J_(CF)=8.0 Hz), 116.7 (d, J_(CF)=22.6 Hz), 62.3, 53.6, 48.3,41.1, 28.4, 23.08, 23.06*, 13.98, 13.96*. * second diastereoisomer. IR(thin film): 3073, 2970, 2876, 2799, 1667, 1652, 1608, 1588, 1532, 1493,1346, 1294, 1235, 1157, 1078, 1059 cm⁻¹. MS (ES-API) m/z: 420.1 (100%,[M+H]⁺, C₂₀H₂₃FN₃O₄S requires 420.1), 442.2 (12%, [M+Na]⁺). mp: 88-90°C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-((4-nitrophenyl)thio)benzamide(DS-1-137)

Following the general procedure A-2 using 4-nitrothiophenol.Purification by chromatography on silica gel (0-15% MeOH in DCM, MeOHcontaining 1% NH₃). Yellow solid (45%). ¹H NMR (400 MHz, CDCl₃) δ 8.66(d, J=1.6 Hz, 1H), 8.35-8.28 (m, 2H), 7.86 (d, J=8.5 Hz, 1H), 7.77-7.71(m, 2H), 7.21 (bs, 1H), 7.02 (d, J=8.5 Hz, 1H), 3.71 (ddd, J=13.8, 7.3,3.0 Hz, 1H), 3.41-3.32 (m, 1H), 3.30-3.23 (m, 1H), 2.92-2.76 (m, 2H),2.40-2.23 (m, 2H), 1.96 (dq, J=12.6, 8.2 Hz, 1H), 1.84-1.58 (m, 3H),1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.6, 148.7, 145.8,139.9, 139.6, 135.6, 133.2, 131.8, 129.4, 125.1, 124.6, 62.3, 53.6,48.3, 40.9, 28.3, 23.1, 14.1. IR (thin film): 3098, 2970, 2876, 2804,1659, 1652, 1607, 1578, 1548, 1520, 1464, 1344, 1294, 1244, 1178, 1109,1047, 1014 cm⁻¹. MS (ES-API) m/z: 431.2 (100%, [M+H]⁺, C₂₀H₂₃N₄O₅Srequires 431.1). mp: 128-129° C.

4-(cyclohexylthio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-139)

Following the general procedure A-2 using cyclohexanethiol. Yellow solid(74%). ¹H NMR (400 MHz, CDCl₃) δ 8.50 (d, J=1.9 Hz, 1H), 7.95 (dd,J=8.5, 1.9 Hz, 1H), 7.51 (d, J=8.5 Hz, 1H), 6.99 (bs, 1H), 3.69 (ddd,J=13.6, 7.2, 2.7 Hz, 1H), 3.37-3.29 (m, 2H), 3.25-3.17 (m, 1H), 2.83(dq, J=12.2, 7.4 Hz, 1H), 2.74-2.67 (m, 1H), 2.32-2.17 (m, 2H),2.12-2.03 (m, 2H), 1.92 (dq, J=12.3, 8.3 Hz, 1H), 1.87-1.80 (m, 2H),1.78-1.56 (m, 4H), 1.53-1.23 (m, 5H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 165.1, 146.4, 140.7, 131.4, 131.1, 127.7, 124.5,62.2, 53.7, 48.2, 44.2, 41.0, 32.5, 28.4, 26.1, 25.7, 23.1, 14.2. IR(thin film): 2969, 2933, 2855, 2800, 1639, 1609, 1539, 1520, 1450, 1328,1288, 1179, 1101, 1052 cm⁻¹. MS (ES-API) m/z: 392.2 (100%, [M+H]⁺,C₂₀H₃₀N₃O₃S requires 392.2). mp: 145-146° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-fluoro-3-nitrobenzamidehydrochloride (DS-1-153)

Following the general procedure B-3 using 4-fluoro-3-nitrobenzoic acidand 2-(aminomethyl)-1-ethylpyrrolidine. White solid (89%). ¹H NMR (400MHz, DMSO-d₆) δ 10.71 (bs, 1H), 9.50 (t, J=5.6 Hz, 1H), 8.69 (dd, J=7.3,2.2 Hz, 1H), 8.43 (ddd, J=8.7, 4.1, 2.3 Hz, 1H), 7.74 (dd, J=11.1, 8.8Hz, 1H), 3.89-3.78 (m, 1H), 3.71-3.60 (m, 2H), 3.55 (dq, J=12.1, 5.6 Hz,1H), 3.50-3.36 (m, 1H), 3.13-3.02 (m, 2H), 2.17-2.06 (m, 1H), 2.03-1.74(m, 3H), 1.30 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.8,156.4 (d, J_(CF)=266.4 Hz), 136.7 (d, J_(CF)=7.8 Hz), 135.1 (d,J_(CF)=10.1 Hz), 130.5 (d, J_(CF)=3.6 Hz), 125.6 (d, J_(CF)=2.0 Hz),118.9 (d, J_(CF)=21.4 Hz), 65.9, 52.6, 48.5, 38.9, 27.1, 21.7, 10.2. IR(thin film): 3249, 3058, 2946, 2655, 2508, 1655, 1618, 1534, 1492, 1352,1316, 1269 cm⁻¹. mp: 161° C.

4-bromo-3-nitrobenzoic acid (DS-1-159)

A 200 mL flask was charged with 4-bromobenzoic acid (9.84 g, 49.0 mmol),nitric acid (70%, 90 mL) and fuming nitric acid (90%, 70 mL), fittedwith a reflux condenser and the resulting suspension was stirred underreflux overnight. The homogeneous yellow solution obtained was cooled to0° C., filtered and the solid washed with cold water (100 mL) to yield awhite powder. Recrystallization from methanol/water afforded the product(10.82 g, 44.0 mmol, 90%) as a microcrystalline white powder. ¹H NMR(400 MHz, DMSO-d₆) δ 13.74 (bs, 1H), 8.39 (s, 1H), 8.09-7.94 (m, 2H).¹³C NMR (100 MHz, DMSO-d₆) δ 165.0, 149.6, 135.4, 133.8, 131.7, 126.0,118.2.

4-((4-chlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-163)

Following the general procedure A-3 using DS-1-153 and4-chlorothiophenol. Purification by chromatography on silica gel (0-20%MeOH in DCM, MeOH containing 1% NH₃). Yellow solid (64%). ¹H NMR (400MHz, CDCl₃) δ 8.64 (d, J=1.9 Hz, 1H), 7.77 (dd, J=8.5, 1.9 Hz, 1H),7.55-7.45 (m, 4H), 7.02 (s, 1H), 6.89 (d, J=8.5 Hz, 1H), 3.68 (ddd,J=13.7, 7.3, 2.7 Hz, 1H), 3.31 (dt, J=13.7, 3.4 Hz, 1H), 3.23-3.17 (m,1H), 2.81 (dq, J=12.2, 7.4 Hz, 1H), 2.70 (bs, 1H), 2.33-2.15 (m, 2H),1.92 (dq, J=12.3, 8.3 Hz, 1H), 1.80-1.54 (m, 3H), 1.11 (t, J=7.2 Hz,3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 144.6, 142.7, 137.3, 137.2,132.1, 131.6, 130.8, 128.9, 128.4, 124.5, 62.2, 53.6, 48.2, 40.9, 28.3,23.1, 14.1. IR (thin film): 2971, 2877, 2810, 1645, 1609, 1548, 1520,1475, 1389, 1338, 1294, 1242, 1093, 1048, 1013 cm⁻¹. MS (ES-API) m/z:420.1 (100%, [M+H]⁺, C₂₀H₂₃ClN₃O₃S requires 420.1). mp: 126° C.

4-bromo-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamidehydrochloride (DS-1-175)

Following the general procedure B-3 using DS-1-159 (8.13 mmol) and2-(aminomethyl)-1-ethylpyrrolidine. White solid (85%). ¹H NMR (400 MHz,DMSO-d₆) δ 10.76 (bs, 1H), 9.52 (t, J=5.6 Hz, 1H), 8.55 (d, J=1.4 Hz1H), 8.17 (dd, J=8.3, 1.6 Hz, 1H), 8.05 (d, J=8.3 Hz, 1H), 3.87-3.78 (m,1H), 3.71-3.60 (m, 2H), 3.54 (dt, J=11.8, 6.0 Hz, 1H), 3.49-3.38 (m,1H), 3.16-3.01 (m, 2H), 2.20-2.06 (m, 1H), 2.03-1.75 (m, 3H), 1.29 (t,J=7.1 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9, 149.5, 135.1, 134.2,132.2, 124.4, 116.6, 65.8, 52.5, 48.5, 38.9, 27.1, 21.6, 10.3. IR (thinfilm): 2684, 1648, 1538, 1470, 1397, 1354, 1311, 1247, 1032 cm⁻¹. MS(ES-API) m/z: 356.1 (100%, [M−Cl]⁺, C₁₄H₁₉BrN₃O₃ requires 356.1). mp:165° C. (dec.).

4-(4-aminophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-177)

Following the general procedure A-3 using DS-1-153 (1 eq),4-aminothiophenol (2 eq) and sodium bicarbonate (3 eq). After overnightreaction, the reaction mixture was diluted with dichloromethane (20 mL)and the aqueous layer was basified to pH˜12 by adding solid potassiumhydroxide. The organic phase was washed with saturated sodiumbicarbonate (2×20 mL) and then extracted with 2M HCl (3×10 mL). Theacidic aqueous phases were basified to pH˜10 with a concentratedpotassium hydroxide solution and extracted with dichloromethane (3×10mL). The organic layers were dried over magnesium sulfate, filtered andevaporated under reduced pressure to afford a red-orange oil.Purification by column chromatography on silica gel (0-25% MeOH in DCM,MeOH containing 1% NH₃). Orange solid (53%). ¹H NMR (400 MHz, CDCl₃) δ8.65 (d, J=1.6 Hz, 1H), 7.77 (dd, J=8.4, 1.5 Hz, 1H), 7.35-7.30 (m, 2H),7.13 (bs, 1H), 6.95 (d, J=8.6 Hz, 1H), 6.79-6.74 (m, 2H), 4.00 (s, 2H),3.69 (ddd, J=14.0, 7.3, 3.4 Hz, 1H), 3.40-3.31 (m, 1H), 3.30-3.23 (m,1H), 2.90-2.74 (m, 2H), 2.37-2.23 (m, 2H), 1.94 (dq, J=11.8, 7.9 Hz,1H), 1.80-1.59 (m, 3H), 1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 165.2, 149.0, 145.4, 144.0, 137.5, 131.2, 131.0, 128.1, 124.5, 116.3,116.3, 62.4, 53.6, 48.3, 41.0, 28.2, 23.0, 13.9. IR (thin film): 2970,2876, 2806, 1652, 1644, 1598, 1548, 1520, 1498, 1463, 1338, 1293, 1240,1178, 1106, 1049, 910, 829, 733 cm⁻¹. MS (ES-API) m/z: 401.1 (55%,[M+H]⁺, C₂₀H₂₅N₄O₃S requires 401.2). mp: 166-168° C. (dec.).

4-((4-(((1-ethylpyrrolidin-2-yl)methyl)carbamoyl)-2-nitrophenyl)thio)benzoicacid hydrochloride (DS-1-179)

A 15 mL pressure tube was charged with the aryl fluoride DS-1-153 (332mg, 1.00 mmol, 1 eq), water (5 mL) and 4-mercaptobenzoic acid (170 mg,1.10 mmol, 1.1 eq). The tube was sealed and the reaction mixture wasstirred at 90° C. for 2 h. A yellow precipitate appeared and thesuspension was stirred overnight at room temperature. The solid wasdissolved by adding 2M sodium hydroxide (50 mL) and the aqueous phasewas washed with dichloromethane (3×50 mL), acidified to pH˜2-3 withconcentrated HCl, washed with dichloromethane (3×50 mL) and concentratedunder reduced pressure to afford a yellow slurry. Toluene (20 mL) wasintroduced and the solvent was evaporated. This process was repeatedtwice to yield a yellow solid that was triturated with 2M HCl (60 mL),filtered, washed with water (20 mL) and diethyl ether (50 mL) and driedunder high vacuum. Trituration with a minimum amount of acetone affordedthe product (180 mg, 0.39 mmol, 39%) as a yellow solid. ¹H NMR (400 MHz,DMSO-d₆) δ 13.15 (s, 1H), 10.54 (s, 1H), 9.39 (s, 1H), 8.77 (d, J=1.9Hz, 1H), 8.13 (dd, J=8.6, 1.8 Hz, 1H), 8.07 (d, J=8.3 Hz, 2H), 7.75 (d,J=8.3 Hz, 2H), 7.04 (d, J=8.5 Hz, 1H), 3.83-3.74 (m, 1H), 3.67-3.58 (m,2H), 3.56-3.49 (m, 1H), 3.45-3.27 (m, 1H), 3.10-3.01 (m, 2H), 2.09 (dt,J=13.2, 6.7 Hz, 1H), 2.02-1.72 (m, 3H), 1.27 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, DMSO-d₆) δ 166.6, 164.2, 145.0, 139.9, 135.5, 135.0, 132.6,132.3, 131.5, 131.0, 129.0, 124.8, 65.9, 52.6, 48.5, 39.9, 27.1, 21.6,10.3. IR (thin film): 2977, 1714, 1694, 1652, 1645, 1634, 1607, 1538,1520, 1506, 1464, 1456, 1394, 1336, 1103, 1047, 1014 cm⁻¹. MS (ES-API)m/z: 430.1 (100%, [M−Cl]⁺, C₂₁H₂₄N₃O₅S requires 430.1). mp: 135-137° C.(dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4′-fluoro-2-nitro-[1,1′-biphenyl]-4-carboxamide(DS-1-181)

Following general procedure C using 4-fluorophenylboronic acid. Lightyellow solid (53%). ¹H NMR (400 MHz, CDCl₃) δ 8.27 (d, J=1.7 Hz, 1H),8.03 (dd, J=8.0, 1.7 Hz, 1H), 7.50-7.46 (m, 1H), 7.31-7.25 (m, 2H),7.14-7.08 (m, 2H), 3.73 (ddd, J=13.8, 7.2, 3.0 Hz, 1H), 3.36 (dt,J=13.8, 3.5 Hz, 1H), 3.29-3.20 (m, 1H), 2.91-2.81 (m, 1H), 2.81-2.75 (m,1H), 2.30 (ddt, J=22.6, 17.0, 8.2 Hz, 2H), 1.95 (dq, J=12.4, 7.9 Hz,1H), 1.82-1.59 (m, 3H), 1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 165.1, 163.1 (d, J_(CF)=248.9 Hz), 149.2, 137.8, 135.1, 132.6 (d,J_(CF)=3.6 Hz), 132.4, 130.6, 129.7 (d, J_(CF)=8.4 Hz), 123.1, 116.0 (d,J_(CF)=21.9 Hz), 62.6, 53.6, 48.4, 401.0, 28.3, 23.1, 13.9. IR (thinfilm): 3075, 2972, 2878, 2812, 1659, 1652, 1645, 1557, 1538, 1532, 1520,1487, 1456, 1356, 1314, 1296, 1229, 1162, 1098, 1008, 911, 837, 734cm⁻¹. MS (ES-API) m/z: 372.2 (100%, [M+H]⁺, C₂₀H₂₃FN₃O₃ requires 372.2).mp: 101° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-3′-fluoro-2-nitro-[1,1′-biphenyl]-4-carboxamide(DS-1-183)

Following general procedure C using 3-fluorophenylboronic acid. Lightbrown wax (90%). ¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J=1.5 Hz, 1H), 8.05(dd, J=8.0, 1.6 Hz, 1H), 7.47 (d, J=8.0 Hz, 1H), 7.40 (td, J=8.0, 5.9Hz, 1H), 7.12 (td, J=8.5, 2.5 Hz, 1H), 7.06 (d, J=7.7 Hz, 1H), 7.02 (dt,J=9.4, 2.0 Hz, 1H), 3.71 (ddd, J=13.9, 7.1, 3.5 Hz, 1H), 3.39 (dt,J=13.8, 3.3 Hz, 1H), 3.31-3.24 (m, 1H), 2.94-2.80 (m, 2H), 2.41-2.28 (m,2H), 1.96 (dq, J=12.3, 7.8 Hz, 1H), 1.85-1.60 (m, 3H), 1.16 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.2, 162.7 (d, J_(CF)=247.4 Hz),149.0, 138.6 (d, J_(CF)=8.0 Hz), 137.6 (d, J_(CF)=2.3 Hz), 135.2, 132.2,130.6, 130.5 (d, J=8.5 Hz), 123.6 (d, J=3.1 Hz), 123.2, 115.7 (d, J=21.1Hz), 115.0 (d, J_(CF)=22.8 Hz), 62.9, 53.6, 48.6, 41.1, 28.2, 23.0,13.6. IR (thin film): 3073, 2973, 2879, 2814, 1660, 1652, 1645, 1614,1588, 1557, 1538, 1532, 1476, 1436, 1357, 1300, 1270, 1188, 1159 cm⁻¹.MS (ES-API) m/z: 372.2 (100%, [M+H]⁺, C₂₀H₂₃FN₃O₃ requires 372.2).

4-((4-bromophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-185)

Following the general procedure A-3 using DS-1-153 and4-bromothiophenol. Purification by trituration with pentane/diethylether 2:1 (3×3 mL). Yellow solid (87%). ¹H NMR (400 MHz, CDCl₃) δ 8.67(d, J=1.8 Hz, 1H), 7.80 (dd, J=8.5, 1.9 Hz, 1H), 7.66-7.62 (m, 2H),7.47-7.43 (m, 2H), 7.20 (bs, 1H), 6.90 (d, J=8.5 Hz, 1H), 3.70 (ddd,J=13.8, 7.4, 3.1 Hz, 1H), 3.38-3.31 (m, 1H), 3.24 (t, J=6.8 Hz, 1H),2.89-2.79 (m, 1H), 2.77 (bs, 1H), 2.36-2.21 (m, 2H), 1.94 (dq, J=12.7,8.3 Hz, 1H), 1.81-1.57 (m, 3H), 1.14 (t, J=7.2 Hz, 3H). ¹³C NMR (100MHz, CDCl₃) δ 165.0, 144.6, 142.4, 137.5, 133.7, 131.9, 131.7, 129.5,128.4, 125.4, 124.7, 62.6, 53.6, 48.4, 40.8, 28.1, 23.1, 13.9. IR (thinfilm): 2968, 1660, 1652, 1644, 1607, 1564, 1548, 1538, 1520, 1470, 1338,1293, 1068, 1049, 1010 cm⁻¹. MS (ES-API) m/z: 464.1 (100%, [M+H]⁺,C₂₀H₂₃BrN₃O₃S requires 464.1). mp: 131-132° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamidehydrochloride (DS-1-191)

In a 5 mL flask, DS-1-033 (110 mg, 0.27 mmol, 1 eq) was suspended inmethanol (2 mL) and a solution of hydrochloric acid in methanol (0.55 M,1 mL, 0.55 mmol, 2 eq) was added dropwise. The resulting yellowhomogeneous solution was concentrated under reduced pressure to affordthe hydrochloride salt (117 mg, 0.27 mmol, 97%) as a yellow powder. 1HNMR (400 MHz, CDCl₃) δ 11.61 (bs, 1H), 9.26 (bs, 1H), 8.95 (s, 1H), 8.16(d, J=8.7 Hz, 1H), 7.57 (dd, J=8.3, 5.3 Hz, 2H), 7.22 (t, J=8.5 Hz, 2H),6.91 (d, J=8.4 Hz, 1H), 3.97-3.80 (m, 3H), 3.78-3.66 (m, 1H), 3.34-3.18(m, 1H), 3.15-2.97 (m, 1H), 3.00-2.88 (m, 1H), 2.32-2.16 (m, 1H),2.14-2.05 (m, 2H), 2.02-1.91 (m, 1H), 1.45 (bs, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 165.1, 164.2 (d, J_(CF)=252.4 Hz), 144.7, 143.7, 138.3 (d,J_(CF)=8.7 Hz), 131.3, 130.2, 128.4, 126.3, 125.3 (d, J_(CF)=3.5 Hz),117.9 (d, J_(CF)=22.1 Hz), 67.8, 54.1, 51.8, 40.1, 27.7, 23.9, 10.8. IR(thin film): 3252, 3061, 2943, 2649, 2505, 2214, 1660, 1652, 1608, 1590,1538, 1520, 1489, 1464, 1398, 1338, 1309, 1227, 1158, 1107, 1092, 1049cm⁻¹. MS (ES-API) m/z: 404.1 (100%, [M−Cl]⁺, C₂₀H₂₃FN₃O₃S requires404.1). mp: 186-187° C. (dec.).

N-(2-((2-aminoethyl)(methyl)amino)ethyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-1-195)

A 50 mL oven-dried flask was charged with2,2′-diamino-N-methyldiethylamine (3.2 mL, 25.00 mmol, 5 eq) anddichloromethane (13 mL) and cooled to −15° C. A solution of the acylchloride DS-1-059 in dichloromethane (15 mL) was added dropwise over 30min and the reaction mixture was slowly warmed to room temperature.After 10 h, the resulting yellow suspension was diluted withdichloromethane (20 mL) and quenched carefully with saturated sodiumbicarbonate (50 mL). The organic layer was washed with saturated sodiumbicarbonate (2×25 mL) and water (25 mL), dried over magnesium sulfateand concentrated under reduced pressure. Purification by columnchromatography on silica gel (0-50% MeOH in DCM, MeOH containing 1% NH₃)afforded the product (408 mg, 1.04 mmol, 21%) as a yellow solid. ¹H NMR(400 MHz, CDCl₃) δ 8.80 (dd, J=4.6, 1.7 Hz, 1H), 8.16-8.10 (m, 1H), 7.90(ddd, J=8.5, 3.5, 1.9 Hz, 1H), 7.57 (ddd, J=8.0, 5.1, 2.5 Hz, 2H), 7.21(td, J=8.6, 2.5 Hz, 2H), 6.83 (dd, J=8.5, 3.7 Hz, 1H), 3.55-3.48 (m,2H), 2.89-2.83 (m, 2H), 2.62 (t, J=5.4 Hz, 2H), 2.53 (q, J=5.9 Hz, 2H),2.30 (s, 3H), 2.14 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.6, 164.1 (d,J_(CF)=252.1 Hz), 144.1, 142.9, 138.2 (d, J_(CF)=8.6 Hz), 132.4, 131.8,127.9, 125.6 (d, J_(CF)=3.3 Hz), 124.5, 117.7 (d, J_(CF)=22.0 Hz), 58.5,54.8, 42.6, 39.2, 38.2. IR (thin film): 3290, 3095, 2950, 2852, 2804,1652, 1645, 1634, 1608, 1590, 1549, 1520, 1490, 1464, 1398, 1338, 1227,1158, 1107, 1092, 1050, 1014 cm⁻¹. MS (ES-API) m/z: 393.1 (100%, [M+H]⁺,C₁₈H₂₂FN₄O₃S requires 393.1). mp: 95-97° C. (dec.).

6-((4-fluorophenyl)thio)nicotinic acid (DS-1-203)

Following the general procedure A-1 using 6-chloronicotinic acid (5.00mmol). White powder (97%). ¹H NMR (400 MHz, DMSO-d₆) δ 13.30 (s, 1H),8.84 (dd, J=2.3, 0.8 Hz, 1H), 8.07 (dd, J=8.4, 2.3 Hz, 1H), 7.73-7.66(m, 2H), 7.42-7.34 (m, 2H), 6.99 (dd, J=8.4, 0.8 Hz, 1H). ¹³C NMR (100MHz, DMSO-d₆) δ 166.0, 165.5 (d, J_(CF)=1.4 Hz), 163.2 (d, J_(CF)=248.2Hz), 150.3, 137.93 (d, J_(CF)=9.1 Hz), 137.88, 124.4 (d, J_(CF)=3.4 Hz),123.0, 119.8, 117.3 (d, J_(CF)=22.1 Hz). IR (thin film): 2363, 1678,1587, 1489, 1421, 1369, 1301, 1281, 1226, 1148, 1104, 1090, 1015 cm⁻¹.MS (ES-API) m/z: 248.0 (100%, [M−H]⁻, C₁₂H₇FNO₂S requires 248.0), 519.1(12%, [2M−2H+Na]⁻). mp: 194° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-6-((4-fluorophenyl)thio)nicotinamide(DS-1-209)

Following the general procedure B-3 using DS-1-203 and2-(aminomethyl)-1-ethylpyrrolidine. Purification by columnchromatography on silica gel (0-50% MeOH in DCM, MeOH containing 1%NH₃). Colorless oil (69%). ¹H NMR (400 MHz, CDCl₃) δ 8.81 (d, J=2.0 Hz,1H), 7.97 (dd, J=8.4, 2.3 Hz, 1H), 7.60-7.55 (m, 2H), 7.48 (bs, 1H),7.18-7.10 (m, 2H), 6.89 (d, J=8.4 Hz, 1H), 3.70 (ddd, J=13.9, 7.2, 4.0Hz, 1H), 3.40 (dt, J=14.0, 3.4 Hz, 1H), 3.34-3.29 (m, 1H), 2.95-2.84 (m,2H), 2.46-2.32 (m, 2H), 1.96 (dq, J=12.4, 7.9 Hz, 1H), 1.85-1.62 (m,3H), 1.16 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.6, 165.3 (d,J_(CF)=1.4 Hz), 163.8 (d, J_(CF)=250.8 Hz), 148.3, 137.8 (d, J_(CF)=8.5Hz), 135.7, 126.1, 125.0 (d, J_(CF)=3.5 Hz), 120.1, 117.2 (d,J_(CF)=22.0 Hz), 63.5, 53.7, 49.0, 40.4, 28.2, 23.2, 13.3. IR (thinfilm): 3289, 3061, 2970, 2876, 2803, 1659, 1652, 1645, 1588, 1549, 1538,1491, 1455, 1360, 1316, 1272, 1225, 1157, 1111, 1092, 1014 cm⁻¹. MS(ES-API) m/z: 360.1 (100%, [M+H]⁺, C₁₉H₂₃FN₃OS requires 360.2).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-(4-fluorophenylsulfonamido)-3-nitrobenzamide(DS-1-213)

A 10 mL oven-dried flask was charged with 4-fluorobenzenesulfonamide(193 mg, 1.10 mmol, 1.1 eq) and dry dimethylformamide (2.8 mL), andpotassium tert-butoxide (129 mg, 1.15 mmol, 1.15 eq) was added at 0° C.The resulting suspension was added to a 15 mL oven-dried pressure tubecontaining the aryl fluoride DS-1-153 (332 mg, 1.00 mmol, 1 eq) andpotassium tert-butoxide (112 mg, 1.00 mmol, 1 eq) in drydimethylformamide (2.5 mL) cooled to 0° C. The pressure tube was sealedand heated to 90° C. After 16 h, the reaction mixture was concentratedunder reduced pressure and the residue taken in dichloromethane (20 mL).The organic phase was washed with 10% lithium chloride (3×10 mL), driedover magnesium sulfate and concentrated under reduced pressure to affordan orange solid. Purification by column chromatography on silica gel(0-50% MeOH in DCM, MeOH containing 1% NH₃) yielded the product (67 mg,0.15 mmol, 15%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.53 (s,1H), 8.44 (bs, 1H), 8.04 (dd, J=8.7, 1.4 Hz, 1H), 7.94-7.88 (m, 2H),7.70 (d, J=8.7 Hz, 1H), 7.18-7.11 (m, 2H), 3.85-3.71 (m, 1H), 3.70-3.60(m, 2H), 3.51-3.43 (m, 1H), 3.18-3.06 (m, 1H), 2.86-2.68 (m, 2H),2.19-2.08 (m, 1H), 2.04-1.91 (m, 2H), 1.91-1.78 (m, 1H), 1.30 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.8, 165.4 (d, J_(CF)=255.8 Hz),139.0, 138.4, 136.0 (d, J_(CF)=3.2 Hz), 133.3, 130.1 (d, J_(CF)=9.5 Hz),127.3, 125.7, 120.5, 116.8 (d, J_(CF)=22.7 Hz), 66.1, 54.0, 50.8, 40.8,28.0, 23.5, 11.8. IR (thin film): 3282, 2983, 2661, 1710, 1652, 1608,1520, 1493, 1354, 1304, 1226, 1127, 1088, 970 cm⁻¹. MS (ES-API) m/z:451.1 (100%, [M+H]⁺, C₂₀H₂₄FN₄O₅S requires 451.1). mp: 94-95° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-hydroxyphenyl)thio)-3-nitrobenzamide(DS-1-217)

A 250 mL pressure flask was charged with the aryl fluoride DS-1-153(3.32 g, 10.0 mmol, 1 eq), water (50 mL), 4-mercaptophenol (1.33 g, 10.5mmol, 1.05 eq) and sodium bicarbonate (1.76 g, 21.0 mmol, 2.1 eq). Theflask was sealed and the reaction mixture was stirred at 90° C. for 4 h.A reddish oil separated and the reaction mixture was stirred overnightat room temperature. The reaction mixture was diluted with saturatedsodium bicarbonate (50 mL), dichloromethane (25 mL) and methanol (25mL). The aqueous layer was extracted with dichloromethane (2×20 mL). Theorganic phase was washed with water (50 mL) and brine (50 mL), driedover magnesium sulfate and evaporated under reduced pressure to affordthe product (3.97 g, 9.9 mmol, 99%) as an orange solid. ¹H NMR (400 MHz,CDCl₃) δ 8.75 (d, J=1.8 Hz, 1H), 7.84 (bs, 1H), 7.82 (dd, J=8.6, 1.7 Hz,1H), 7.40-7.37 (m, 2H), 6.91-6.88 (m, 3H), 5.43 (bs, 1H), 3.87-3.77 (m,1H), 3.55-3.45 (m, 1H), 3.41-3.32 (m, 1H), 3.03-2.91 (m, 2H), 2.54-2.39(m, 2H), 2.07-1.96 (m, 1H), 1.90-1.69 (m, 3H), 1.20 (t, J=7.2 Hz, 3H).¹³C NMR (100 MHz, CDCl₃) δ 165.8, 160.0, 145.4, 144.2, 138.0, 131.1,130.7, 128.2, 125.1, 118.4, 118.4, 64.0, 53.7, 49.1, 40.6, 27.7, 23.0,13.1. IR (thin film): 3289, 3094, 2972, 1652, 1607, 1520, 1496, 1455,1338, 1279, 1167, 1106, 1049 cm⁻¹. MS (ES-API) m/z: 402.1 (100%, [M+H]⁺,C₂₀H₂₄N₃O₄S requires 402.1). mp: 93-94° C. (dec.).

3-cyano-4-fluorobenzoic acid (DS-1-219)

A 1 L flask was charged with 5-fluoro-2-formylbenzonitrile (10.0 g, 67.0mmol, 1 eq), water (100 mL) and tert-butanol (450 mL). After 10 min,sodium phosphate monobasic hydrate (21.8 g, 158.0 mmol, 2.36 eq) andsodium chlorite (28.0 g, 248.0 mmol, 3.7 eq) were introducedsuccessively in one portion under vigorous stirring. The colorlesssuspension rapidly turned orange and an orange gas formed. After 18 h,the reaction mixture has turned yellow and concentrated hydrochloricacid was added at 0° C. until the white solid was entirely dissolved.The tert-butanol was evaporated under reduced pressure in awell-ventilated fume-hood (caution: a yellow gas escaped) and theresulting suspension was diluted to 500 mL with water, filtered andwashed with water (1 L). The white solid obtained was dissolved in ethylacetate (80 mL), washed with brine (2×30 mL), dried over magnesiumsulfate, filtered and concentrated under reduced pressure to yield theproduct (7.14 g, 43.2 mmol, 65%) as a white powder. ¹H NMR (400 MHz,DMSO-d₆) δ 13.57 (s, 1H), 8.35 (dd, J=6.3, 2.2 Hz, 1H), 8.26 (ddd,J=8.8, 5.3, 2.2 Hz, 1H), 7.61 (t, J=9.0 Hz, 1H). ¹³C NMR (100 MHz,DMSO-d₆) δ 164.9, 164.9 (d, J_(CF)=261.5 Hz), 137.0 (d, J_(CF)=10.2 Hz),135.2 (d, J_(CF)=1.2 Hz), 128.5 (d, J_(CF)=3.3 Hz), 117.1 (d,J_(CF)=20.3 Hz), 113.3, 101.0 (d, J_(CF)=16.1 Hz).

3-cyano-N-((1-ethylpyrrolidin-2-yl)methyl)-4-fluorobenzamidehydrochloride (DS-1-223)

Following the general procedure B-3 using 3-cyano-4-fluorobenzoic acid(DS-1-219) and 2-(aminomethyl)-1-ethylpyrrolidine. Beige solid (93%). ¹HNMR (400 MHz, DMSO-d₆) δ 10.63 (bs, 1H), 9.39 (t, J=5.6 Hz, 1H), 8.51(dd, J=6.2, 2.3 Hz, 1H), 8.35 (ddd, J=8.8, 5.2, 2.3 Hz, 1H), 7.68 (t,J=9.0 Hz, 1H), 3.86-3.77 (m, 1H), 3.70-3.49 (m, 3H), 3.50-3.34 (m, 1H),3.13-3.01 (m, 2H), 2.18-2.05 (m, 1H), 2.01-1.75 (m, 3H), 1.29 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.0, 164.0 (d, J_(CF)=260.3 Hz),135.4 (d, J_(CF)=9.8 Hz), 133.3, 131.0 (d, J_(CF)=3.3 Hz), 116.9 (d,J_(CF)=20.1 Hz), 113.5, 100.4 (d, J_(CF)=15.9 Hz), 65.9, 52.6, 48.5,38.7, 27.0, 21.6, 10.3. IR (thin film): 3249, 2947, 2652, 1661, 1611,1588, 1549, 1496, 1318, 1271, 1106 cm⁻¹. MS (ES-API) m/z: 276.1 (100%,[M+H]⁺, C₁₅H₁₉FN₃O requires 276.1). mp: 164-166° C. (dec.).

3-amino-N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)benzamide(DS-1-225)

A 50 mL flask equipped with a reflux condenser was charged with DS-1-033(403 mg, 1.00 mmol, 1 eq), iron powder (343 mg, 6.15 mmol, 6.15 eq),ammonium chloride (350 mg, 6.55 mmol, 6.55 eq), water (6.6 mL) andethanol (13.3 mL). The reaction mixture was heated to 90° C. and stirredvigorously. After 1 h, the reaction mixture was cooled to roomtemperature and diluted with ethyl acetate (50 mL). The organic phasewas washed with water (2×25 mL) and brine (25 mL), dried over magnesiumsulfate and concentrated under reduced pressure to afford the product(360 mg, 0.96 mmol, 96%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ7.38 (d, J=8.0 Hz, 1H), 7.27 (d, J=1.8 Hz, 1H), 7.15-7.05 (m, 2H), 7.02(dd, J=8.0, 1.8 Hz, 2H), 6.96-6.89 (m, 2H), 4.46 (s, 2H), 3.68 (ddd,J=13.7, 7.4, 3.2 Hz, 1H), 3.33-3.26 (m, 1H), 3.22 (ddd, J=9.5, 6.3, 3.5Hz, 1H), 2.84 (dq, J=12.2, 7.4 Hz, 1H), 2.78-2.68 (m, 1H), 2.34-2.17 (m,2H), 1.90 (dq, J=12.2, 8.2 Hz, 1H), 1.78-1.57 (m, 3H), 1.12 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 167.2, 161.1 (d, J_(CF)=245.8 Hz),148.3, 136.4, 135.8, 130.3 (d, J_(CF)=3.1 Hz), 129.4 (d, J_(CF)=7.9 Hz),118.3, 115.8 (d, J_(CF)=22.1 Hz), 115.7, 114.0, 62.5, 53.2, 48.1, 40.9,27.9, 22.6, 13.4. IR (thin film): 2970, 2877, 2809, 1711, 1652, 1634,1615, 1591, 1558, 1538, 1489, 1424, 1226, 1157 cm⁻¹. MS (ES-API) m/z:374.2 (100%, [M+H]⁺, C₂₀H₂₅FN₃OS requires 374.2).

3-cyano-N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)benzamide(DS-1-227)

Following the general procedure A-3 using DS-1-223 and4-fluorothiophenol. White solid (88%). ¹H NMR (400 MHz, CDCl₃) δ 8.07(d, J=1.6 Hz, 1H), 8.22 (dd, J=8.5, 1.5 Hz, 1H), 7.60-7.50 (m, 2H),7.20-7.13 (m, 2H), 6.95 (d, J=8.5 Hz, 1H), 3.70 (ddd, J=13.9, 7.3, 3.3Hz, 1H), 3.39-3.32 (m, 1H), 3.30-3.25 (m, 1H), 2.92-2.76 (m, 2H),2.40-2.23 (m, 2H), 2.04 (bs, 1H), 1.95 (dq, J=12.4, 8.1 Hz, 1H),1.84-1.58 (m, 3H), 1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ163.9, 163.0 (d, J_(CF)=248.2 Hz), 144.9, 136.7 (d, J_(CF)=8.8 Hz),132.8, 132.7, 132.4, 128.2, 125.1 (d, J_(CF)=3.4 Hz), 117.5 (d,J_(CF)=22.2 Hz), 116.3, 110.1, 62.6, 53.2, 48.2, 43.4, 28.7, 22.4, 13.8.IR (thin film): 2970, 2876, 2803, 2226, 1645, 1592, 1538, 1490, 1463,1312, 1227, 1157, 1054, 836 cm⁻¹. MS (ES-API) m/z: 384.2 (100%, [M+H]⁺,C₂₁H₂₃FN₃OS requires 384.2). mp: 79° C. (dec.).

N¹-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)isophthalamide(DS-1-231-M)

In a 25 mL flask, DS-1-227 (100 mg, 0.261 mmol, 1 eq) was dissolved inethanol (5 mL). Aqueous 2M sodium hydroxide (5 mL) was introduced andthe resulting white suspension was heated to reflux. After 1 h, thereaction mixture was extracted with dichloromethane (3×10 mL) at roomtemperature. The organic layers were dried over anhydrous magnesiumsulfate and concentrated under reduced pressure to afford the crudeproduct as a yellow oil. Purification by column chromatography on silicagel (0-50% MeOH in DCM, MeOH containing 1% NH₃) afforded the product (50mg, 0.12 mmol, 48%) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ8.17 (d, J=1.6 Hz, 1H), 7.60 (dd, J=8.4, 1.7 Hz, 1H), 7.48 (dd, J=8.6,5.3 Hz, 2H), 7.37 (bs, 1H), 7.10 (t, J=8.6 Hz, 2H), 7.00 (bs, 1H), 6.83(d, J=8.4 Hz, 1H), 3.59 (ddd, J=13.5, 6.9, 3.5 Hz, 1H), 3.45 (bs, 1H),3.30 (dt, J=13.6, 3.7 Hz, 1H), 3.16-3.09 (m, 1H), 2.81 (dq, J=14.7, 7.3Hz, 1H), 2.69 (dt, J=9.9, 4.2 Hz, 1H), 2.30-2.13 (m, 2H), 1.88 (dq,J=12.1, 8.1 Hz, 1H), 1.76-1.50 (m, 3H), 1.08 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 169.8, 166.5, 163.4 (d, J_(CF)=250.4 Hz), 143.8,137.3 (d, J_(CF)=8.4 Hz), 131.8, 131.0, 129.0, 127.7, 127.33, 127.29 (d,J_(CF)=3.7 Hz), 117.1 (d, J_(CF)=21.9 Hz), 62.5, 53.5, 48.5, 41.4, 28.2,22.9, 13.8. IR (thin film): 2970, 2876, 2808, 1668, 1652, 1644, 1591,1538, 1490, 1469, 1408, 1378, 1310, 1225, 1156, 1046, 834 cm⁻¹. MS(ES-API) m/z: 402.2 (100%, [M+H]⁺, C₂₁H₂₅FN₃O₂S requires 402.2). mp:75-76° C.

N-(2-(diethylamino)ethyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamidehydrochloride (DS-1-239)

Following the general procedure D using DS-1-043 (0.13 mmol). Yellowsolid (100%). ¹H NMR (400 MHz, DMSO-d₆) δ 10.47 (bs, 1H), 9.31 (t, J=5.4Hz, 1H), 8.76 (d, J=1.8 Hz, 1H), 8.12 (dd, J=8.6, 1.9 Hz, 1H), 7.72 (dd,J=8.7, 5.4 Hz, 2H), 7.44 (t, J=8.8 Hz, 2H), 6.89 (d, J=8.6 Hz, 1H), 3.66(q, J=6.0 Hz, 2H), 3.22 (t, J=5.9 Hz, 2H), 3.16 (q, J=6.9 Hz, 4H), 1.22(t, J=7.2 Hz, 6H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.0, 163.5 (d,J_(CF)=249.1 Hz), 144.1, 141.6 (d, J_(CF)=1.2 Hz), 138.3 (d, J_(CF)=8.9Hz), 132.6, 131.1, 127.9, 125.3 (d, J_(CF)=3.3 Hz), 124.8, 117.8 (d,J_(CF)=22.1 Hz), 49.6, 46.5, 34.3, 8.3. IR (thin film): 3246, 2980,2644, 1652, 1608, 1590, 1540, 1520, 1489, 1472, 1398, 1338, 1312, 1224,1158, 1048, 838 cm⁻¹. MS (ES-API) m/z: 392.1 (100%, [M−Cl]⁺,C₁₉H₂₃FN₃O₃S requires 392.1). mp: 170-171° C.

4-((4-fluorophenyl)thio)-N-(2-morpholinoethyl)-3-nitrobenzamidehydrochloride (DS-1-241)

Following the general procedure D using DS-1-089 (0.12 mmol). Yellowsolid (100%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.12 (bs, 1H), 9.27 (t, J=5.2Hz, 1H), 8.77 (d, J=1.5 Hz, 1H), 8.12 (dd, J=8.5, 1.5 Hz, 1H), 7.72 (dd,J=8.3, 5.4 Hz, 2H), 7.44 (t, J=8.7 Hz, 2H), 6.89 (d, J=8.5 Hz, 1H), 3.95(d, J=11.3 Hz, 2H), 3.87-3.78 (m, 2H), 3.70 (d, J=5.4 Hz, 2H), 3.52 (d,J=12.0 Hz, 2H), 3.30 (d, J=5.1 Hz, 2H), 3.15-3.04 (m, 2H). ¹³C NMR (100MHz, DMSO-d₆) δ 164.0, 163.5 (d, J_(CF)=249.2 Hz), 144.1, 141.5, 138.3(d, J_(CF)=8.9 Hz), 132.7, 131.2, 127.8, 125.3 (d, J_(CF)=3.2 Hz),124.8, 117.8 (d, J_(CF)=22.1 Hz), 63.1, 55.2, 51.1, 33.8. IR (thinfilm): 1652, 1608, 1590, 1548, 1520, 1489, 1456, 1338, 1226, 1158, 1104,1048, 836 cm⁻¹. MS (ES-API) m/z: 406.1 (52%, [M−Cl]⁺, C₁₉H₂₁FN₃O₄Srequires 406.1), 169.1 (100%). mp: 184-185° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-((4-(prop-2-yn-1-yloxy)phenyl)thio)benzamide(DS-1-261)

A 5 mL flask was charged with DS-1-217 (201 mg, 0.50 mmol, 1 eq),dimethylformamide (2.5 mL), potassium carbonate (138 mg, 1.00 mmol, 2eq) and propargyl bromide (80% wt. in toluene, 50 μL, 0.45 mmol, 0.9eq). The resulting suspension was stirred overnight and concentratedunder reduced pressure to afford a black residue that was partitionedbetween dichloromethane (10 mL) and saturated sodium bicarbonate (10mL). The aqueous phase was extracted with dichloromethane (2×10 mL) andthe organic layers were washed with water (30 mL) and brine (30 mL),dried over anhydrous magnesium sulfate and concentrated under reducedpressure to furnish a brown oil. Purification by column chromatographyon silica gel (0-50% MeOH in DCM, MeOH containing 1% NH₃) yielded theproduct (38 mg, 0.09 mmol, 19%) as a brown oil. ¹H NMR (400 MHz, CDCl₃)δ 8.68 (d, J=1.9 Hz, 1H), 7.80 (dd, J=8.6, 1.9 Hz, 1H), 7.54-7.49 (m,2H), 7.34 (bs, 1H), 7.13-7.08 (m, 2H), 6.90 (d, J=8.5 Hz, 1H) 4.78 (d,J=2.4 Hz, 2H), 3.71 (ddd, J=13.8, 7.2, 3.4 Hz, 1H), 3.37 (dt, J=13.9,3.6 Hz, 1H), 3.29-3.25 (m, 1H), 2.94-2.78 (m, 2H), 2.60 (t, J=2.4 Hz,1H), 2.43-2.24 (m, 2H), 1.95 (dq, J=12.4, 8.1 Hz, 1H), 1.83-1.60 (m,3H), 1.16 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 159.3,144.2, 144.1, 137.6, 131.3, 131.2, 128.2, 124.6, 121.5, 116.7, 77.8,76.3, 62.8, 55.9, 53.6, 48.5, 40.7, 28.1, 23.0, 13.6. IR (thin film):2972, 1652, 1645, 1608, 1548, 1519, 1493, 1338, 1290, 1242, 1176, 1108,1049, 1022 cm⁻¹. MS (ES-API) m/z: 440.2 (100%, [M+H]⁺, C₂₃H₂₆N₃O₄Srequires 440.2).

Ethyl2-((4-(((1-ethylpyrrolidin-2-yl)methyl)carbamoyl)-2-nitrophenyl)thio)-5-fluorobenzoate(DS-1-265)

Following the general procedure A-3 using DS-1-153 (5.18 mmol) and ethyl5-fluoro-2-mercaptobenzoate (5.44 mmol). Purification by washing thecrude solid with 1M sodium hydroxide (50 mL), water (50 mL) and hexanes(50 mL), and dried under vacuum. Yellow solid (94%). ¹H NMR (400 MHz,CDCl₃) δ 8.65 (d, J=1.6 Hz, 1H), 7.78 (dd, J=8.5, 1.7 Hz, 1H), 7.67-7.61(m, 2H), 7.29 (td, J=8.3, 2.9 Hz, 1H), 7.08 (bs, 1H), 6.89 (d, J=8.5 Hz,1H), 4.25 (q, J=7.1 Hz, 2H), 3.69 (ddd, J=13.7, 7.3, 2.7 Hz, 1H), 3.31(dt, J=13.8, 3.1 Hz, 1H), 3.24-3.18 (m, 1H), 2.82 (dq, J=14.7, 7.4 Hz,1H), 2.72 (bs, 1H), 2.34-2.16 (m, 2H), 1.92 (dq, J=12.3, 8.2 Hz, 1H),1.81-1.53 (m, 3H), 1.19 (t, J=7.1 Hz, 3H), 1.12 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.2 (d, J_(CF)=2.4 Hz), 165.0, 163.5 (d, J_(CF)253.8 Hz), 144.8, 142.3 (d, J_(CF)=0.9 Hz), 140.0 (d, J_(CF)=8.1 Hz),139.0 (d, J_(CF)=7.8 Hz), 132.1, 131.4, 129.3, 126.0 (d, J_(CF)=3.8 Hz),124.4, 120.0 (d, J_(CF)=21.4 Hz), 118.7 (d, J_(CF)=24.0 Hz), 62.3, 62.2,53.6, 48.2, 40.9, 28.2, 23.0, 14.04, 14.02. IR (thin film): 3073, 2971,2876, 2803, 1732, 1646, 1607, 1578, 1520, 1465, 1339, 1293, 1271, 1248,1200, 1103, 1044 cm⁻¹. MS (ES-API) m/z: 476.2 (100%, [M+H]⁺,C₂₃H₂₇FN₃O₅S requires 476.2). mp: 56° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(quinolin-8-ylthio)benzamide(DS-1-269)

Following the general procedure A-3 using DS-1-153, 8-quinolinethiolhydrochloride and sodium bicarbonate (3 eq). Yellow solid (96%). ¹H NMR(400 MHz, CDCl₃) δ 8.95 (d, J=2.6 Hz, 1H), 8.66 (s, 1H), 8.29 (d, J=8.1Hz, 1H), 8.14 (d, J=6.9 Hz, 1H), 8.05 (d, J=8.1 Hz, 1H), 7.66 (t, J=7.6Hz, 1H), 7.59 (d, J=8.1 Hz, 1H), 7.52 (dd, J=8.0, 3.9 Hz, 1H), 6.99 (bs,1H), 6.68 (d, J=8.5 Hz, 1H), 3.65 (ddd, J=13.1, 6.9, 2.3 Hz, 1H), 3.28(d, J=13.3 Hz, 1H), 3.21-3.15 (m, 1H), 2.80 (dq, J=14.4, 7.4 Hz, 1H),2.69 (bs, 1H), 2.31-2.15 (m, 2H), 1.95-1.84 (m, 1H), 1.78-1.53 (m, 3H),1.10 (t, J=7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.2, 151.8, 148.0,144.8, 142.7, 138.4, 137.1, 131.6, 131.5, 131.2, 130.4, 129.5, 129.3,127.2, 124.3, 122.3, 62.3, 53.6, 48.2, 41.0, 28.3, 23.1, 14.1. IR (thinfilm): 2970, 2803, 1654, 1606, 1548, 1519, 1491, 1457, 1337, 1293, 1050,828, 790 cm⁻¹. MS (ES-API) m/z: 437.2 (35%, [M+H]⁺, C₂₃H₂₅N₄O₃S requires437.2), 219.1 (100%). mp: 203° C. (dec.).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(p-tolylthio)benzamide(DS-1-271)

Following the general procedure A-3 using DS-1-153 and p-thiocresol.Pale yellow solid (95%). ¹H NMR (400 MHz, CDCl₃) δ 8.67 (s, 1H), 7.78(dd, J=8.5, 1.2 Hz, 1H), 7.46 (d, J=7.8 Hz, 2H), 7.31 (d, J=7.8 Hz, 2H),7.19 (bs, 1H), 6.90 (d, J=8.5 Hz, 1H). 3.71 (ddd, J=13.4, 7.2, 2.4 Hz,1H), 3.31 (d, J=13.8 Hz, 1H), 3.20 (t, J=7.0 Hz, 1H), 2.84 (dq, J=14.6,7.3 Hz, 1H), 2.71 (bs, 1H), 2.44 (s, 3H), 2.34-2.15 (m, 2H), 1.98-1.84(m, 1H), 1.79-1.56 (m, 2H), 1.12 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 165.0, 144.3, 143.9, 140.9, 135.9, 131.4, 131.3, 131.1, 128.3,126.5, 124.4, 62.4, 53.5, 48.2, 40.7, 28.0, 22.9, 21.4, 13.9. IR (thinfilm): 2968, 2875, 2802, 1639, 1608, 1546, 1521, 1461, 1337, 1293, 1049,812 cm⁻¹. MS (ES-API) m/z: 400.2 (100%, [M+H]⁺, C₂₁H₂₆N₃O₃S requires400.2). mp: 83-85° C.

4-((4-fluorophenyl)thio)-3-nitro-N-((tetrahydrofuran-2-yl)methyl)benzamide(DS-1-275)

In a 4 mL oven-dried vial, was suspended the acyl chloride DS-1-059 (312mg, 1.00 mmol, 1 eq) in dry dichloromethane (1 mL). Pyridine (0.16 mL,2.0 mmol, 2 eq) and tetrahydrofurfurylamine (0.12 mL, 1.2 mmol, 1.2 eq)were added sequentially dropwise. After 48 h, the yellow suspension wasfiltered and the solid washed with diethyl ether (10 mL) and dissolvedin dichloromethane (20 mL). The organic phase was washed with 10%hydrochloric acid (6×15 mL), saturated sodium bicarbonate (20 mL), water(20 mL) and brine (20 mL), dried over anhydrous magnesium sulfate andconcentrated under reduced pressure to afford the product (257 mg, 0.68mmol, 68%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J=1.9Hz, 1H), 7.79 (dd, J=8.5, 2.0 Hz, 1H), 7.63-7.53 (m, 2H), 7.25-7.18 (m,2H), 6.86 (d, J=8.5 Hz, 1H), 6.54 (bs, 1H), 4.05 (qd, J=7.2, 3.2 Hz,1H), 3.88 (dt, J=8.3, 6.7 Hz, 1H), 3.84-3.74 (m, 2H), 3.30 (ddd, J=13.7,7.9, 4.5 Hz, 1H), 2.09-1.99 (m, 1H), 1.93 (p, J=6.8 Hz, 2H), 1.64-1.53(m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 164.7, 164.2 (d, J_(CF)=252.3 Hz),144.3, 143.4 (d, J_(CF)=1.6 Hz), 138.3 (d, J_(CF)=8.6 Hz), 131.9, 131.5,128.2, 125.6 (d, J_(CF)=3.6 Hz), 124.3, 117.8 (d, J_(CF)=22.0 Hz), 77.6,68.2, 44.1, 28.9, 26.0. IR (thin film): 2870, 1639, 1607, 1590, 1547,1520, 1491, 1338, 1293, 1226, 1158, 1078, 1049, 836 cm⁻¹. MS (ES-API)m/z: 377.1 (100%, [M+H]⁺, C₁₈H₁₈FN₂O₄S requires 377.1), 775.2 (37%,[2M+Na]⁺). mp: 172° C.

(4-((4-fluorophenyl)thio)-3-nitrophenyl)(1-methylhexahydropyrrolo[1,2-a]pyrazin-2(1H)-yl)methanone(DS-1-279)

In a 4 mL oven-dried vial, was suspended the acyl chloride (312 mg, 1.00mmol, 1 eq) in dry dichloromethane (1 mL). Dimethylaminopyridine (2 mg,0.02 mmol, 2 mol %) and 1-methyloctahydropyrrolo[1,2-a]pyrazine (168 mg,1.20 mmol, 1.2 eq) were added sequentially. After 48 h, the yellowsuspension was filtered and the solid washed with diethyl ether (10 mL)and dissolved in dichloromethane (20 mL). The organic phase was washedwith 1M sodium hydroxide (3×20 mL), water (20 mL) and brine (20 mL),dried over anhydrous magnesium sulfate and concentrated under reducedpressure to afford a yellow solid. Purification by column chromatographyon silica gel (0-50% MeOH in DCM, MeOH containing 1% NH₃) yielded theproduct (169 mg, 0.41 mmol, 41%) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.29 (d, J=1.8 Hz, 1H), 7.60-7.54 (m, 2H), 7.40 (dd, J=8.4, 1.9Hz, 1H), 7.23-7.16 (m, 2H), 6.83 (d, J=8.4 Hz, 1H), 4.20-4.13 (m, 1H),3.81 (d, J=13.8 Hz, 1H), 3.35 (ddd, J=13.8, 11.1, 4.1 Hz, 1H), 2.90(ddd, J=10.4, 8.2, 1.8 Hz, 1H), 2.74-2.58 (m, 3H), 2.50 (dt, J=11.0, 3.5Hz, 1H), 1.98-1.86 (m, 1H), 1.85-1.64 (m, 2H), 1.66-1.51 (m, 1H), 1.42(d, J=6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.4, 164.2 (d,J_(CF)=252.2 Hz), 144.3, 141.6 (d, J_(CF)=1.6 Hz), 138.3 (d, J_(CF)=8.7Hz), 133.4, 132.1, 128.3, 125.6 (d, J_(CF)=3.5 Hz), 124.6, 117.8 (d,J_(CF)=22.0 Hz), 64.4, 54.6, 52.0, 48.1, 40.6, 26.3, 21.5, 18.2. IR(thin film): 2968, 2878, 2809, 1633, 1608, 1590, 1547, 1520, 1491, 1428,1337, 1291, 1227, 1157, 1050, 836 cm⁻¹. MS (ES-API) m/z: 416.1 (100%,[M+H]⁺, C₂₁H₂₃FN₃O₃S requires 416.1). mp: 67-68° C.

4-(4-fluorophenyl)thio)-N-(3-morpholinopropyl)-3-nitrobenzamide(DS-1-283)

In a 4 mL oven-dried vial, was suspended the acyl chloride DS-1-059 (312mg, 1.00 mmol, 1 eq) in dry dichloromethane (1 mL).Dimethylaminopyridine (1 mg, 0.01 mmol, 1 mol %) and3-morpholinopropylamine (0.17 mL, 1.20 mmol, 1.2 eq) were addedsequentially. After 48 h, the yellow suspension was filtered and thesolid washed with diethyl ether (20 mL) and dissolved in dichloromethane(40 mL). The organic phase was washed with 1M sodium hydroxide (3×15mL), brine (20 mL), dried over anhydrous magnesium sulfate andconcentrated under reduced pressure to afford the product (363 mg, 0.87mmol, 87%) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (s, 1H),8.35 (bs, 1H), 7.90 (dd, J=8.6, 1.9 Hz, 1H), 7.63-7.53 (m, 2H),7.25-7.18 (m, 2H), 6.89 (d, J=8.6 Hz, 1H) 3.75 (s, 4H), 3.59 (q, J=5.8Hz, 2H), 2.66-2.50 (m, 6H), 1.84 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ164.5, 164.2 (d, J_(CF)=252.3 Hz), 144.0, 143.4, 138.2 (d, J_(CF)=8.7Hz), 132.3, 131.9, 128.2, 125.5 (d, J_(CF)=3.5 Hz), 123.7, 117.8 (d,J_(CF)=22.0 Hz), 66.9, 58.9, 54.0, 41.1, 23.9. IR (thin film): 3286,3076, 2956, 2855, 2814, 1640, 1608, 1590, 1547, 1520, 1491, 1462, 1337,1292, 1223, 1158, 1117, 1048, 836 cm⁻¹. MS (ES-API) m/z: 420.1 (100%,[M+H]⁺, C₂₀H₂₃FN₃O₄S requires 420.1). mp: 144° C.

(1-ethylpyrrolidin-2-yl)methyl 4-((4-fluorophenyl)thio)-3-nitrobenzoate(DS-1-287)

In a 10 mL oven-dried flask, were suspended the acyl chloride DS-1-059(623 mg, 2.00 mmol, 2 eq) and dimethylaminopyridine (12 mg, 0.10 mmol,0.1 eq) in dry dichloromethane (5 mL).1-Ethyl-2-hydroxymethylpyrrolidine (0.135 mL, 1.00 mmol, 1 eq) was addeddropwise at 0° C. After 24 h, the reaction mixture was diluted withdichloromethane (20 mL) and 1M sodium hydroxide (40 mL). The aqueousphase was extracted with dichloromethane (3×10 mL). The combined organiclayers were washed with 1M sodium hydroxide (5×10 mL), water (30 mL) andbrine (30 mL), dried over anhydrous magnesium sulfate and concentratedunder reduced pressure to afford the product (212 mg, 0.52 mmol, 52%) asa brown oil. ¹H NMR (400 MHz, CDCl₃) δ 8.84 (d, J=1.8 Hz, 1H), 7.94 (dd,J=8.6, 1.8 Hz, 1H), 7.62-7.52 (m, 2H), 7.24-7.17 (m, 2H), 6.86 (d, J=8.6Hz, 1H), 4.31 (dd, J=11.0, 5.3 Hz, 1H), 4.21 (dd, J=11.0, 6.4 Hz, 1H),3.15 (ddd, J=9.2, 6.5, 2.8 Hz, 1H), 2.91 (dq, J=12.0, 7.4 Hz, 1H), 2.81(dq, J=8.5, 5.6 Hz, 1H), 2.41 (dq, J=12.1, 6.6 Hz, 1H), 2.25 (td, J=9.2,7.5 Hz, 1H), 1.96 (dq, J=12.2, 8.4 Hz, 1H), 1.84-1.72 (m, 2H), 1.74-1.62(m, 1H), 1.11 (d, J=14.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.4,164.3 (d, J_(CF)=252.5 Hz), 145.2, 144.4, 138.3 (d, J_(CF)=8.7 Hz),133.6, 128.0, 127.6, 127.2, 125.4, 117.9 (d, J_(CF)=22.0 Hz), 68.6,62.1, 54.0, 49.5, 28.7, 23.2, 14.0. IR (thin film): 3100, 2970, 2877,2798, 1723, 1608, 1591, 1556, 1526, 1491, 1385, 1339, 1306, 1287, 1234,1158, 1132, 1105, 1051, 837, 750 cm⁻¹. MS (ES-API) m/z: 405.1 (100%,[M+H]⁺, C₂₀H₂₂FN₂O₄S requires 405.1), 391.1 (6%, [M−CH₃+H]⁺).

4-((3,4-dichlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-291)

Following the general procedure A-3 using DS-1-153 and3,4-dichlorothiophenol. Yellow solid (56%). ¹H NMR (400 MHz, CDCl₃) δ8.71 (s, 1H), 7.87 (d, J=8.0 Hz, 1H), 7.70 (d, J=1.8 Hz, 1H), 7.59 (d,J=8.3 Hz, 1H), 7.42 (dd, J=8.3, 1.8 Hz, 1H), 6.94 (d, J=8.5 Hz, 1H),3.72 (ddd, J=13.1, 6.7, 3.0 Hz, 1H), 3.43-3.35 (m, 1H), 3.32-3.25 (m,1H), 2.94-2.81 (m, 2H), 2.43-2.27 (m, 2H), 1.96 (dq, J=16.7, 7.6 Hz,1H), 1.83-1.61 (m, 4H), 1.17 (t, J=6.9 Hz, 3H). ¹³C NMR (100 MHz,DMSO-d₆) δ 163.6, 144.5, 139.6, 136.6, 135.4, 133.6, 132.8, 132.7,132.4, 132.3, 131.0, 128.8, 124.4, 62.5, 53.2, 48.3, 43.5, 28.7, 22.4,13.9. IR (thin film): 2969, 2799, 1659, 1652, 1634, 1607, 1548, 1520,1454, 1338, 1293, 1247, 1141, 1048, 1033 cm⁻¹. MS (ES-API) m/z: 454.1(100%, [M+H]⁺, C₂₀H₂₂Cl₂N₃O₃S requires 454.1). mp: 146-148° C. (dec.).

4-((3-chlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-295)

Following the general procedure A-3 using DS-1-153 and3-chlorothiophenol. Yellow solid (80%). ¹H NMR (400 MHz, CDCl₃) δ 8.68(d, J=1.3 Hz, 1H), (dd, J=8.4, 1.1 Hz, 1H), 7.60 (s, 1H), 7.53-7.42 (m,3H), 7.19 (bs, 1H), 6.93 (d, J=8.5 Hz, 1H), 3.71 (ddd, J=13.8, 7.3, 3.1Hz, 1H), 3.39-3.32 (m, 1H), 3.29-3.22 (m, 1H), 2.90-2.75 (m, 2H),2.39-2.21 (m, 2H), 1.94 (dq, J=12.3, 8.2 Hz, 1H), 1.84-1.56 (m, 3H),1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 144.7, 142.2,135.9, 135.6, 134.1, 132.4, 132.1, 131.8, 131.5, 130.8, 128.5, 124.6,62.6, 53.6, 48.3, 40.8, 28.0, 23.0, 14.0. IR (thin film): 3072, 2969,2875, 2799, 1644, 1607, 1564, 1548, 1520, 1463, 1338, 1293, 1242, 1116,1048, 782 cm⁻¹. MS (ES-API) m/z: 420.1 (100%, [M+H]⁺, C₂₀H₂₃ClN₃O₃Srequires 420.1). mp: 73-74° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-fluoro-3-(methylsulfonyl)benzamidehydrochloride (DS-1-297)

Following the general procedure B-3 using4-fluoro-3-(methylsulfonyl)benzoic acid and2-(aminomethyl)-1-ethylpyrrolidine. Brown solid (100%). ¹H NMR (400 MHz,DMSO-d₆/H₂O) δ 10.21 (bs, 1H), 9.32 (t, J=5.1 Hz, 1H), 8.38 (dd, J=6.8,2.0 Hz, 2H), 7.70 (t, J=9.6 Hz, 1H), 3.80-3.72 (m, 1H), 3.70-3.65 (m,1H), 3.66-3.53 (m, 2H), 3.51-3.39 (m, 1H), 3.38 (s, 3H), 3.07 (bs, 2H),2.12 (dt, J=12.8, 6.4 Hz, 1H), 1.96 (dt, J=12.7, 5.7 Hz, 1H), 1.93-1.74(m, 2H), 1.28 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆/H₂O) δ 164.4,160.5 (d, J_(CF)=258.3 Hz), 135.3 (d, J_(CF)=9.7 Hz), 130.6 (d,J_(CF)=3.4 Hz), 129.0, 128.5 (d, J_(CF)=15.8 Hz), 117.8 (d, J_(CF)=22.1Hz), 65.9, 52.6, 48.5, 43.6, 39.0, 27.2, 21.7, 10.3. IR (thin film):3243, 2933, 2638, 1652, 1602, 1557, 1486, 1455, 1393, 1315, 1261, 1146,1066, 963, 844, 776 cm⁻¹. MS (ES-API) m/z: 329.1 (100%, [M−Cl]⁺,C₁₅H₂₂FN₂O₃S requires 329.1).

4-((3,4-dimethylphenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-1-299)

Following the general procedure A-3 using DS-1-153 and3,4-dimethylthiophenol. Yellow solid (93%). ¹H NMR (400 MHz, CDCl₃) δ8.67 (s, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.36-7.23 (m, 3H), 6.92 (d, J=8.6Hz, 1H), 3.71 (ddd, J=13.5, 6.9, 3.1 Hz, 1H), 3.37 (d, J=13.8 Hz, 1H),3.27 (bs, 1H), 3.32-3.21 (m, 1H), 2.92-2.77 (m, 1H), 2.39-2.19 (m, 8H),1.94 (dt, J=17.0, 8.3 Hz, 1H), 1.83-1.59 (m, 4H), 1.15 (t, J=7.0 Hz,3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.1, 144.1, 142.1, 139.1, 139.2,136.4, 133.2, 132.5, 131.7, 128.1, 126.2, 124.6, 62.9, 53.4, 48.5, 43.3,28.7, 22.5, 19.5, 19.4, 13.8. IR (thin film): 2968, 2805, 1634, 1608,1557, 1538, 1520, 1470, 1348, 1294, 1250, 1115, 1050 cm⁻¹. MS (ES-API)m/z: 414.2 (100%, [M+H]⁺, C₂₂H₂₈N₃O₃S requires 414.2). mp: 91-92° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-(methylsulfonyl)benzamide(DS-1-301)

A 15 mL pressure tube was charged with the aryl fluoride DS-1-297 (332mg, 1.00 mmol, 1 eq), water (5 mL), 4-fluorothiophenol (0.12 mL, 1.10mmol, 1.1 eq) and sodium bicarbonate (176 mg, 2.10 mmol, 2.1 eq). Thetube was sealed and the reaction mixture was stirred at 90° C. for 2 hand then overnight at room temperature. The reaction mixture was dilutedwith water (20 mL) and dichloromethane (20 mL). The aqueous phase wasextracted with dichloromethane (3×10 mL) and the combined organic layerswere washed with water (20 mL) and brine (20 mL), dried over anhydrousmagnesium sulfate and concentrated under reduced pressure to afford thecrude product as a brown oil. Purification by column chromatography onsilica gel (0-50% MeOH in DCM, MeOH containing 1% NH₃) yielded theproduct (241 mg, 0.55 mmol, 55%) as a white solid. ¹H NMR (400 MHz,CDCl₃) δ 8.44 (s, 1H), 7.86 (d, J=8.3 Hz, 1H), 7.61-7.51 (m, 2H),7.22-7.15 (m, 2H), 6.95 (d, J=8.4 Hz, 1H), 3.70 (ddd, J=13.8, 7.2, 3.5Hz, 1H), 3.35 (s, 3H), 3.40-3.24 (m, 2H), 2.93-2.77 (m, 2H), 2.41-2.24(m, 2H), 1.95 (dq, J=16.7, 8.0 Hz, 1H), 1.83-1.59 (m, 4H), 1.16 (t,J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.5, 164.0 (d, J_(CF)=252.0Hz), 144.1 (d, J_(CF)=1.3 Hz), 137.8 (d, J_(CF)=8.6 Hz), 136.6, 132.4,132.3, 128.8, 128.4, 125.5 (d, J_(CF)=3.6 Hz), 117.7 (d, J_(CF)=22.1Hz), 62.5, 53.6, 48.4, 41.9, 41.0, 28.3, 23.1, 14.0. IR (thin film):3063, 2970, 2877, 2803, 1652, 1634, 1592, 1532, 1490, 1455, 1312, 1227,1142, 1038, 959, 837, 737 cm⁻¹. MS (ES-API) m/z: 437.1 (100%, [M+H]⁺,C₂₁H₂₆FN₂O₃S₂ requires 437.1). mp: 82-83° C.

2-(2-(ethylamino)ethyl)isoindoline-1,3-dione hydrochloride (DS-1-303)

In a 200 mL flask, N-ethyl ethylenediamine (5.3 mL, 50.0 mmol, 1 eq) washeated to 100° C. and phthalimide (7.36 g, 50.0 mmol, 1 eq) was addedover 15 min under vigorous stirring. Gaseous ammonia instantaneouslyevolved from the reaction mixture. After 3 h, the resulting yellow oilwas heated to 130° C. After 2 h, the heating bath was removed andethanol (100 mL) was added in one portion to the hot reaction mixture,immediately followed by 5-6N HCl in isopropanol (12 mL) dropwise. Thereaction mixture clogged upon cooling and was cooled in an ice bath. Thesolid was filtered, washed with cold ethanol (100 mL) until the filtratewas colorless, and dried under high vacuum to afford the product (8.07g, 31.7 mmol, 63%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.86(bs, 2H), 7.93-7.84 (m, 4H), 3.89 (t, J=5.9 Hz, 2H), 3.22-3.15 (m, 2H),3.02-2.92 (m, 2H), 1.17 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ167.9, 134.3, 132.0, 123.0, 44.1, 41.7, 33.9, 10.8.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(m-tolylthio)benzamide(DS-1-305)

Following the general procedure A-3 using DS-1-153 and3-methylthiophenol. Yellow solid (92%). ¹H NMR (400 MHz, CDCl₃) δ 8.67(s, 1H), 7.79 (d, J=8.2 Hz, 1H), 7.42-7.37 (m, 3H), 7.38-7.28 (m, 1H),6.93 (d, J=8.5 Hz, 1H), 3.71 (ddd, J=13.8, 7.2, 3.3 Hz, 1H), 3.37 (bd,J=13.0 Hz, 1H), 3.27 (bs, 1H), 2.86 (dq, J=14.7, 7.2 Hz, 2H), 2.41 (s,3H), 2.38-2.22 (m, 2H), 1.95 (dq, J=16.9, 8.2 Hz, 1H), 1.83-1.59 (m,4H), 1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 144.4,143.7, 140.5, 136.5, 133.0, 131.5, 131.4, 130.2, 129.9, 128.5, 124.5,62.5, 53.6, 48.3, 40.8, 28.1, 23.0, 21.4, 14.0. IR (thin film): 3060,2969, 2875, 2799, 1644, 1607, 1548, 1520, 1464, 1337, 1293, 1242, 1107,1049 cm⁻¹. MS (ES-API) m/z: 400.2 (100%, [M+H]⁺, C₂₁H₂₆N₃O₃S requires400.2).

2-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)isoindoline-1,3-dione (DS-1-307)

A 100 mL oven-dried flask was charged with DS-1-303 (2.55 g, 10.00 mmol,1 eq), dry acetonitrile (40 mL) and potassium carbonate (1.38 g, 10.00mmol, 1 eq). Propargyl bromide (80% wt in toluene, 1.1 mL, 10.00 mmol, 1eq) was added dropwise and the reaction mixture was stirred vigorouslyfor 40 h. The solid was filtered and the filtrate concentrated underreduced pressure to afford the crude product as a yellow solid.Purification by column chromatography on silica gel (0-5% MeOH in DCM,MeOH containing 1% NH₃) yielded the product (1.71 g, 6.68 mmol, 67%) asa white powder. ¹H NMR (400 MHz, CDCl₃) δ 7.84-7.79 (m, 2H), 7.74-7.64(m, 2H), 3.77 (t, J=6.5 Hz, 2H), 3.47 (d, J=2.4 Hz, 2H), 2.79 (t, J=6.5Hz, 2H), 2.56 (q, J=7.1 Hz, 2H), 2.15 (t, J=2.4 Hz, 1H), 0.96 (t, J=7.1Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 168.5, 133.9, 132.3, 123.3, 78.6,73.0, 50.7, 47.5, 41.5, 36.0, 12.9. IR (thin film): 3273, 2971, 2941,2828, 1772, 1713, 1468, 1433, 1398, 1358, 1326, 1190, 1172, 1156, 1098,1087, 1030, 720 cm⁻¹. MS (ES-API) m/z: 257.1 (100%, [M+H]⁺, C₁₅H₁₇N₂O₂requires 257.1).

3-cyano-N-((1-ethylpyrrolidin-2-yl)methyl)-4-(4-fluorophenoxy)benzamide(DS-1-311)

A 15 mL pressure tube was charged with the aryl fluoride DS-1-223 (182mg, 0.58 mmol, 1 eq), water (3 mL), 4-fluorophenol (72 mg, 0.64 mmol,1.1 eq) and sodium bicarbonate (103 mg, 1.23 mmol, 2.1 eq). The tube wassealed and the reaction mixture was stirred at 90° C. for 2 h and thenovernight at room temperature. The reaction mixture was diluted withwater (10 mL) and dichloromethane (10 mL). The aqueous phase wasextracted with dichloromethane (2×10 mL) and the combined organic layerswere washed with brine (20 mL), dried over anhydrous magnesium sulfateand concentrated under reduced pressure to afford the crude product as abrown oil. Purification by column chromatography on silica gel (0-5%MeOH in DCM, MeOH containing 1% NH₃) yielded the product (101 mg, 0.28mmol, 47%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.11 (d, J=2.2Hz, 1H), 7.93 (dd, J=8.9, 2.3 Hz, 1H), 7.17-7.08 (m, 4H), 6.82 (d, J=8.8Hz, 1H), 3.70 (ddd, J=13.8, 7.4, 3.0 Hz, 1H), 3.33 (ddd, J=13.8, 4.0,2.7 Hz, 1H), 3.24 (td, J=6.9, 2.4 Hz, 1H), 2.85 (dq, J=12.1, 7.3 Hz,1H), 2.75 (ddd, J=10.7, 6.7, 3.7 Hz, 1H), 2.44 (bs, 1H), 2.35-2.20 (m,2H), 1.99-1.89 (m, 1H), 1.83-1.57 (m, 3H), 1.13 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.1, 162.2 (d, J_(CF)=0.8 Hz), 160.3 (d,J_(CF)=245.1 Hz), 150.1 (d, J_(CF)=2.8 Hz), 133.3, 133.1, 129.5, 122.3(d, J_(CF)=8.5 Hz), 117.2 (d, J_(CF)=23.6 Hz), 115.7, 115.4, 103.3,62.5, 53.7, 48.3, 40.7, 28.2, 23.1, 14.0. IR (thin film): 3075, 2970,2936, 2876, 2802, 2234, 1660, 1652, 1645, 1608, 1548, 1538, 1505, 1486,1268, 1228, 1191, 1147, 1091, 852 cm⁻¹. MS (ES-API) m/z: 368.2 (100%,[M+H]⁺, C₂₁H₂₃FN₃O₂ requires 368.2).

3-cyano-4-fluoro-N-(2-morpholinoethyl)benzamide hydrochloride (DS-2-025)

Following the general procedure B-3 using 3-cyano-4-fluorobenzoic acidand 4-(2-aminoethyl)morpholine. White solid (92%). ¹H NMR (400 MHz,DMSO-d₆) δ 11.01 (bs, 1H), 9.21 (bs, 1H), 8.50 (dd, J=6.2, 2.2 Hz, 1H),8.38-8.28 (m, 1H), 7.67 (t, J=9.0 Hz, 1H), 4.01-3.92 (m, 2H), 3.89-3.78(m, 2H), 3.70 (q, J=5.7 Hz, 2H), 3.53 (d, J=12.1 Hz, 2H), 3.37-3.28 (m,2H), 3.17-3.06 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9 (d,J_(CF)=260.1 Hz), 163.8, 135.5 (d, J_(CF)=9.7 Hz), 133.4, 131.3 (d,J_(CF)=3.3 Hz), 116.8 (d, J_(CF)=20.0 Hz), 113.6, 100.3 (d, J_(CF)=15.8Hz), 63.1, 55.3, 51.1, 33.8. IR (thin film): 1652, 1538, 1495, 1102cm⁻¹. MS (ES-API) m/z: 278.1 (100%, [M−Cl]⁺, C₁₄H₁₇FN₃O₂ requires278.1).

3-cyano-4-((4-fluorophenyl)thio)-N-(2-morpholinoethyl)benzamide(DS-2-027)

Following the general procedure A-3 using DS-2-025 and4-fluorothiophenol. White solid (90%). ¹H NMR (400 MHz, CDCl₃) δ 8.06(d, J=1.6 Hz, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.59-7.52 (m, 2H), 7.21-7.13(m, 2H), 6.95 (d, J=8.5 Hz, 1H), 3.78 (bs, 4H), 3.58 (q, J=5.3 Hz, 2H),2.72-2.55 (m, 6H), 1.83 (bs, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 164.7,163.8 (d, J_(CF)=251.8 Hz), 147.5 (d, J_(CF)=1.3 Hz), 137.3 (d,J_(CF)=8.7 Hz), 132.1 (2C), 131.3, 127.5, 124.7 (d, J_(CF)=3.5 Hz),117.5 (d, J_(CF)=22.2 Hz), 116.1, 110.7, 66.8, 56.7, 53.3, 36.1. IR(thin film): 3065, 2955, 2893, 2856, 2814, 2226, 1641, 1596, 1542, 1490,1461, 1309, 1227, 1157, 1144, 1117, 1054, 836 cm⁻¹. MS (ES-API) m/z:386.1 (100%, [M+H]⁺, C₂₀H₂₁FN₃O₂S requires 386.1).

N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(DS-2-035)

A 50 mL flask was charged with DS-1-307 (768 mg, 3.00 mmol, 1 eq) andethanol (20 mL). Anhydrous hydrazine (0.28 mL, 9.00 mmol, 3 eq) wasadded dropwise and the homogeneous solution was stirred 60 h. Theresulting white precipitate was filtered and washed with ethanol (3×10mL). The colorless filtrate was concentrated under reduced pressure toafford the crude diamine as a yellow oil.

A 10 mL oven-dried flask was charged with the acyl chloride DS-1-059(826 mg, 2.65 mmol, 1 eq), dry dichloromethane (1.8 mL) and4-dimethylaminopyridine (3 mg, 0.03 mmol, 1 mol %) and cooled to 0° C. Asolution of the crude diamine in dry dichloromethane (2 mL) was addeddropwise over 10 min and the reaction mixture was slowly warmed to roomtemperature. After 16 h, the reaction mixture was diluted with 1M sodiumhydroxide (30 mL) and dichloromethane (50 mL). The organic phase waswashed with 1M sodium hydroxide (30 mL), water (30 mL) and brine (30mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to afford the crude product as a yellow solid.Purification by column chromatography on silica gel (0-20% MeOH in DCM,MeOH containing 1% NH₃) yielded the product (679 mg, 1.69 mmol, 64%) asa yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.69 (s, 1H), 7.86 (d, J=9.4Hz, 1H), 7.60-7.55 (m, 2H), 7.21 (t, J=8.6 Hz, 2H), 6.87 (d, J=8.5 Hz,1H), 3.62-3.49 (m, 4H), 2.89 (bs, 2H), 2.73 (bs, 2H), 2.32 (s, 1H), 1.67(bs, 1H), 1.16 (t, J=6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.6,164.2 (d, J_(CF)=252.3 Hz), 144.3, 143.3 (d, J_(CF)=1.6 Hz), 138.3 (d,J_(CF)=8.7 Hz), 131.8, 131.7, 128.2, 125.6 (d, J_(CF)=3.6 Hz), 124.3,117.8 (d, J_(CF)=22.0 Hz), 78.4, 73.4, 51.6, 47.5, 41.5, 37.4, 12.8. IR(thin film): 3094, 2973, 2840, 1652, 1644, 1634, 1608, 1590, 1549, 1520,1491, 1464, 1338, 1294, 1226, 1184, 1157, 1109, 1089, 1049, 839 cm⁻¹. MS(ES-API) m/z: 402.1 (100%, [M+H]⁺, C₂₀H₂₁FN₃O₃S requires 402.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-3,4-difluorobenzamide (DS-2-039)

Following the general procedure B-3 using 3,4-difluorobenzoic acid and2-(aminomethyl)-1-ethylpyrrolidine. Off-white solid (80%). ¹H NMR (400MHz, CDCl₃) δ 11.79 (s, 1H), 9.09 (t, J=5.9 Hz, 1H), 7.98-7.90 (m, 2H),7.28-7.21 (m, 1H), 3.93-3.80 (m, 3H), 3.77-3.65 (m, 1H), 3.29-3.14 (m,1H), 3.11-2.96 (m, 1H), 2.97-2.87 (m, 1H), 2.27-1.95 (m, 4H), 1.45 (t,J=7.3 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 164.5, 151.6 (dd,J_(CF)=241.8, 12.7 Hz), 149.1 (dd, J_(CF)=237.4, 12.7 Hz), 131.1 (dd,J_(CF)=4.8, 3.6 Hz), 124.9 (dd, J_(CF)=7.4, 3.4 Hz), 117.7 (d,J_(CF)=17.6 Hz), 116.8 (d, J_(CF)=18.5 Hz), 66.0, 52.6, 48.6, 38.8,27.0, 21.8, 10.3. IR (thin film): 3260, 3042, 2947, 2651, 2507, 1660,1652, 1606, 1557, 1513, 1506, 1428, 1300, 1283, 1203, 1108, 776 cm⁻¹. MS(ES-API) m/z: 269.1 (100%, [M+H]⁺, C₁₄H₁₉F₂N₂O requires 269.1).

Ethyl 4-iodo-3-nitrobenzoate (DS-2-041)

A 25 mL flask was charged with ethyl 4-iodobenzoate (5.52 g, 20.0 mmol,1 eq) and sulfuric acid (5 mL) and cooled to 0° C. An ice-cooled mixtureof fuming nitric acid (1.9 mL) and sulfuric acid (2.7 mL) was addeddropwise over 30 min under vigorous stirring with a Pasteur pipette. Theresulting black homogeneous mixture was warmed to room temperature andstirred 5 h to afford a yellow suspension that was poured onto ice (100g). The yellow precipitate was filtered, dissolved in ethyl acetate (100mL), washed with saturated sodium carbonate (2×100 mL) and brine (100mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to yield the crude product as a yellow powder.Purification by recrystallization from ethanol at reflux afforded theproduct (4.09 g, 12.7 mmol, 65%). ¹H NMR (400 MHz, CDCl₃) δ 8.45 (d,J=1.9 Hz, 1H), 8.14 (d, J=8.2 Hz, 1H), 7.89 (dd, J=8.2, 2.0 Hz, 1H),4.42 (q, J=7.1 Hz, 2H), 1.42 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 164.2, 153.3, 142.4, 133.6, 132.0, 126.1, 92.1, 62.2, 14.4.

3-cyano-4-(4-fluorophenoxy)-N-(2-morpholinoethyl)benzamide (DS-2-045)

Following the general procedure A-4 using the aryl fluoride DS-2-025 and4-fluorophenol. Purification by column chromatography on silica gel(0-20% MeOH in DCM, MeOH containing 1% NH₃). White solid (74%). ¹H NMR(400 MHz, CDCl₃) δ 8.10 (d, J=1.2 Hz, 1H), 7.93 (d, J=8.8 Hz, 1H),7.19-7.07 (m, 4H), 6.87 (bs, 1H), 6.83 (d, J=8.8 Hz, 1H), 3.77 (bs, 4H),3.58 (q, J=4.9 Hz, 2H), 2.71-2.50 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ164.8, 162.3, 160.3 (d, J_(CF)=245.3 Hz), 150.0 (d, J_(CF)=2.8 Hz),133.4, 133.0, 129.3, 122.3 (d, J_(CF)=8.5 Hz), 117.2 (d, J_(CF)=23.6Hz), 115.7, 115.4, 103.2, 66.9, 57.0, 53.4, 36.2. IR (thin film): 3076,2942, 2857, 2817, 2233, 1659, 1652, 1644, 1609, 1548, 1505, 1486, 1311,1269, 1226, 1191, 1146, 1118, 1011, 852 cm⁻¹. MS (ES-API) m/z: 370.2(100%, [M+H]⁺, C₂₀H₂₁FN₃O₃ requires 370.2).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)(hydroxy)methyl)-3-nitrobenzamide(DS-2-051)

An oven-dried 25 mL flask was charged with ethyl 4-iodo-3-nitrobenzoate(642 mg, 2.00 mmol, 1 eq) and evacuated and backfilled with argon threetimes. Dry tetrahydrofuran (5 mL) was introduced and the flask wascooled to −40° C. Phenylmagnesium chloride (2M in tetrahydrofuran, 1.1mL, 2.20 mmol, 1.1 eq) was added dropwise over 10 min and thehomogeneous solution progressively turned from yellow to dark grey.After 5 min, 4-fluorobenzaldehyde (0.26 mL, 2.40 mmol, 1.2 eq) wasintroduced dropwise over 5 min and the reaction mixture gradually turneddark red. After 30 min, the cooling bath was removed and the solutionwas stirred at room temperature for 1 h. Saturated ammonium chloride (2mL) was added and the reaction mixture was then poured into water (25mL). The aqueous phase was extracted with ethyl acetate (3×20 mL) andthe combined organic layers were washed with brine (50 mL), dried overanhydrous magnesium sulfate and concentrated under reduced pressure toafford the crude product as a brown oil. Purification by columnchromatography on silica gel (0-100% ethyl acetate in hexanes) yieldedthe product as a yellow oil that was used in the next step withoutfurther purification.

A 10 mL flask was charged with the ethyl ester previously prepared and2-(aminomethyl)-1-ethylpyrrolidine (2.8 mL, 20.00 mmol, 10 eq). Theyellow solution immediately turned red and potassium cyanide wasintroduced (7 mg, 0.20 mmol, 0.1 eq). After 5 days, the reaction mixturewas loaded onto silica gel and purified by column chromatography (0-20%MeOH in DCM, MeOH containing 1% NH₃) to yield the product (140 mg, 0.35mmol, 17% in two steps) as an orange solid in a 1:1 mixture of twodiastereoisomers. ¹H NMR (400 MHz, CDCl₃) δ 8.28 (dd, J=3.5, 1.6 Hz,1H), 7.91 (ddd, J=8.0, 2.9, 1.7 Hz, 1H), 7.69 (dd, J=14.7, 8.2 Hz, 1H),7.54-7.47 (m, 1H), 7.31-7.24 (m, 2H), 6.98 (t, J=8.6 Hz, 2H), 6.44 (d,J=5.6 Hz, 1H), 6.08 (bs, 1H), 3.78 (dtd, J=11.4, 8.1, 3.3 Hz, 1H), 3.33(dq, J=11.1, 3.2 Hz, 1H), 2.93 (dt, J=21.7, 7.6 Hz, 1H), 2.88-2.73 (m,1H), 2.69 (bs, 1H), 2.27-2.08 (m, 2H), 1.88 (dt, J=13.8, 7.4 Hz, 1H),1.76-1.54 (m, 3H), 1.04 (dt, J=13.0, 7.2 Hz, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 165.8, 165.7, 162.49 (d, J_(CF)=247.1 Hz), 162.48 (d,J_(CF)=247.2 Hz), 148.02, 147.99, 141.4 (2C), 137.91 (d, J_(CF)=3.3 Hz),137.87 (d, J_(CF)=3.3 Hz), 135.33, 135.28, 131.2, 131.1, 129.07 (2C),129.02 (d, J_(CF)=8.8 Hz, 2C), 128.96 (d, J_(CF)=8.8 Hz, 2C), 123.9(2C), 115.7 (d, J_(CF)=21.5 Hz, 4C), 70.1, 70.0, 63.3 (2C), 53.5, 53.4,48.5 (2C), 40.3, 40.2, 27.33, 27.28, 22.8, 22.6, 13.4, 13.3. IR (thinfilm): 3077, 2973, 2879, 2815, 1660, 1652, 1645, 1604, 1564, 1538, 1506,1354, 1300, 1224, 1181, 1158, 1099, 1045, 842, 814, 738 cm⁻¹. MS(ES-API) m/z: 402.2 (100%, [M+H]⁺, C₂₁H₂₅FN₃O₄ requires 402.2).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-fluoro-4-((4-fluorophenyl)thio)benzamide(DS-2-053)

A 15 mL pressure tube was charged with the aryl fluoride DS-2-039 (305mg, 1.00 mmol, 1 eq), water (5 mL), 4-fluorothiophenol (0.12 mL, 1.10mmol, 1.1 eq) and sodium bicarbonate (176 mg, 2.10 mmol, 2.1 eq). Thetube was sealed and the reaction mixture was stirred at 90° C. for 2 hand then overnight at room temperature. The reaction mixture was dilutedwith 1M sodium hydroxide (10 mL) and dichloromethane (10 mL). Theaqueous phase was extracted with dichloromethane (3×5 mL) and thecombined organic layers were washed with brine (20 mL), dried overanhydrous magnesium sulfate and concentrated under reduced pressure toafford the crude product as a yellow oil. Purification by columnchromatography on silica gel (0-20% MeOH in DCM, MeOH containing 1% NH₃)yielded the product (50 mg, 0.13 mmol, 13%) as a yellow oil. ¹H NMR (400MHz, CDCl₃) δ 7.52 (dd, J=10.2, 1.7 Hz, 1H), 7.50-7.45 (m, 2H), 7.41(dd, J=8.2, 1.7 Hz, 1H), 7.13-7.07 (m, 2H), 7.00-6.95 (m, 1H), 6.94 (bs,1H), 3.68 (ddd, J=13.8, 7.4, 2.8 Hz, 1H), 3.29 (ddd, J=13.7, 4.1, 2.8Hz, 1H), 3.24-3.17 (m, 1H), 2.82 (dq, J=12.2, 7.3 Hz, 1H), 2.74-2.67 (m,1H), 2.32-2.17 (m, 2H), 1.91 (dq, J=12.2, 8.2 Hz, 1H), 1.80-1.56 (m,3H), 1.11 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.8 (d,J_(CF)=2.1 Hz), 163.2 (d, J_(CF)=249.9 Hz), 159.3 (d, J_(CF)=246.9 Hz),136.0 (d, J_(CF)=8.4 Hz), 134.57 (d, J_(CF)=6.4 Hz), 129.8 (d,J_(CF)=1.8 Hz), 129.2 (d, J_(CF)=16.6 Hz), 126.3 (d, J_(CF)=3.3 Hz),122.7 (d, J_(CF)=3.4 Hz), 116.9 (d, J_(CF)=22.1 Hz), 114.5 (d,J_(CF)=23.2 Hz), 62.3, 53.5, 48.1, 40.6, 28.1, 23.0, 14.0. IR (thinfilm): 3068, 2970, 2876, 2800, 1644, 1607, 1590, 1557, 1520, 1490, 1292,1225, 1157, 1090, 1059, 1014, 834 cm⁻¹. MS (ES-API) m/z: 377.1 (100%,[M+H]⁺, C₂₀H₂₃F₂N₂OS requires 377.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitrobenzenesulfonamide(DS-2-055)

A 25 mL flask equipped with a reflux condenser was charged withchlorosulfonic acid (5.2 mL, 78.0 mmol, 2.2 eq) and heated to 65° C.1-Fluoro-2-nitrobenzene (3.7 mL, 35.0 mmol, 1 eq) was added dropwise.The resulting brown mixture was then heated to 100° C. After 14 h, thecooled reaction mixture was poured onto ice (50 g) and the aqueous phasewas extracted with dichloromethane (2×50 mL). The combined organiclayers were washed with saturated sodium bicarbonate (2×50 mL) and brine(50 mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to yield the sulfonyl chloride (5.06 g, 21.1 mmol, 60%)as a brown liquid that was used in the next step without furtherpurification.

In a 100 mL flask, the sulfonyl chloride was dissolved indichloromethane (21 mL) and cooled to 0° C.2-(Aminomethyl)-1-ethylpyrrolidine (2.9 mL, 21.0 mmol, 1 eq) was addeddropwise over 5 min and the reaction mixture was allowed to warm to roomtemperature overnight. After 18 h, the resulting yellow precipitate wasfiltered and washed with diethyl ether (100 mL) to afford the crudesulfonamide as a hygroscopic yellow powder that was used in the nextstep without further purification.

A 15 mL pressure tube was charged with the crude sulfonamide (368 mg,1.00 mmol, 1 eq), water (5 mL), 4-fluorothiophenol (0.12 mL, 1.10 mmol,1.1 eq) and sodium bicarbonate (176 mg, 2.10 mmol, 2.1 eq). The tube wassealed and the reaction mixture was stirred at 90° C. for 2 h and thenovernight at room temperature. The reaction mixture was diluted withsaturated sodium bicarbonate (10 mL) and dichloromethane (10 mL). Theaqueous phase was extracted with dichloromethane (2×10 mL) and thecombined organic layers were washed with sodium bicarbonate (2×10 mL)and brine (20 mL), dried over anhydrous magnesium sulfate andconcentrated under reduced pressure to afford the crude product as anorange oil. Purification by column chromatography on silica gel (0-20%MeOH in DCM, MeOH containing 1% NH₃) yielded the product (223 mg, 0.51mmol, 25% in three steps) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ8.67 (d, J=2.0 Hz, 1H), 7.75 (dd, J=8.6, 2.0 Hz, 1H), 7.60-7.55 (m, 2H),7.25-7.19 (m, 2H), 6.92 (d, J=8.6 Hz, 1H), 4.20 (bs, 1H), 3.11-3.05 (m,1H), 2.97 (dd, J=11.8, 2.6 Hz, 1H), 2.89 (dd, J=11.8, 4.3 Hz, 1H),2.60-2.53 (m, 1H), 2.53-2.44 (m, 1H), 2.18-2.07 (m, 2H), 1.89-1.78 (m,1H), 1.73-1.54 (m, 3H), 0.96 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 164.3 (d, J_(CF)=253.0 Hz), 145.1 (d, J_(CF)=1.5 Hz), 144.1, 138.3 (d,J_(CF)=8.7 Hz), 137.3, 131.0, 128.7, 124.9 (d, J_(CF)=3.6 Hz), 124.8,118.0 (d, J_(CF)=22.1 Hz), 61.8, 53.4, 47.9, 44.1, 28.2, 23.1, 13.9. IR(thin film): 3098, 2971, 2877, 2804, 1593, 1554, 1520, 1491, 1455, 1393,1338, 1291, 1227, 1171, 1157, 1102, 1048, 1014, 942, 890, 837 cm⁻¹. MS(ES-API) m/z: 440.1 (100%, [M+H]⁺, C₁₉H₂₃FN₃O₄S₂ requires 440.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-(4-fluorobenzoyl)-3-nitrobenzamide(DS-2-057)

A 5 mL flask was charged with the alcohol DS-2-051 (50 mg, 0.13 mmol, 1eq), manganese dioxide (109 mg, 1.25 mmol, 9.6 eq) and drydichloromethane (2.5 mL). The resulting black suspension was stirred atroom temperature. After 6 h, TLC analysis indicated incompleteconversion of the starting material and more manganese dioxide (109 mg,1.25 mmol, 9.6 eq) was added. After 14 h, the alcohol was fully consumedand the reaction mixture was filtered through Celite, washing withdichloromethane until the filtrate was colorless. The filtrate wasconcentrated under reduced pressure to afford the crude product as ayellow oil. Purification by column chromatography on silica gel (0-20%MeOH in DCM, MeOH containing 1% NH₃) yielded the product (47 mg, 0.12mmol, 94%) as a yellow oil. 1H NMR (400 MHz, CDCl₃) δ 8.67 (d, J=1.5 Hz,1H), 8.23 (dd, J=7.8, 1.6 Hz, 1H), 7.78-7.72 (m, 2H), 7.53 (bs, 1H),7.53 (d, J=7.8 Hz, 1H), 7.15-7.08 (m, 2H), 3.76 (ddd, J=13.9, 7.2, 3.3Hz, 1H), 3.43-3.36 (m, 1H), 3.25 (ddd, J=9.6, 7.0, 2.8 Hz, 1H),2.94-2.85 (m, 1H), 2.86-2.76 (m, 1H), 2.39-2.23 (m, 2H), 2.03-1.89 (m,1H), 1.85-1.60 (m, 3H), 1.15 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 191.4, 166.3 (d, J_(CF)=257.0 Hz), 164.6, 146.7, 138.1, 137.4, 132.7,132.1 (d, J_(CF)=2.9 Hz), 132.0 (d, J_(CF)=9.6 Hz), 129.3, 123.5, 116.3(d, J_(CF)=22.2 Hz), 62.8, 53.6, 48.5, 41.0, 28.1, 23.1, 13.8. IR (thinfilm): 3075, 2971, 2877, 2803, 1668, 1598, 1538, 1506, 1412, 1349, 1299,1281, 1241, 1152, 938, 852 cm⁻¹. MS (ES-API) m/z: 400.2 (100%, [M+H]⁺,C₂₁H₂₃FN₃O₄ requires 400.2).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-N-methyl-3-nitrobenzamide(DS-2-061)

A 10 mL flask was charged with sodium hydride (95% purity, 48 mg, 2.00mmol, 2 eq) and dry tetrahydrofuran (3 mL) and cooled to 0° C. Asolution of the secondary amide DS-1-033 (403 mg, 1.00 mmol, 1 eq) indry tetrahydrofuran (2 mL) was added dropwise and the yellow mixture wasstirred 1 h at 0° C. and 0.5 h at room temperature. A solution ofiodomethane (0.11 mL, 1.20 mmol, 1.2 eq) in dry tetrahydrofuran (1.2 mL)was introduced dropwise to the yellow suspension. After 2 h, theconversion was complete by TLC analysis and the homogeneous reactionmixture was diluted with water (10 mL). The aqueous phase was extractedwith ethyl acetate (3×10 mL) and the combined organic layers were driedover anhydrous magnesium sulfate and concentrated under reduced pressureto afford the crude product as an orange oil. Purification by columnchromatography on silica gel (0-20% MeOH in DCM, MeOH containing 1% NH₃)yielded the product (183 mg, 0.44 mmol, 44%) as an orange oil in a 1:1.4mixture of two rotamers. ¹H NMR (400 MHz, CDCl₃) δ 8.64* (s, 0.52H),8.28^(†) (s, 0.38H), 7.59-7.54 (m, 2H), 7.48-7.39 (m, 1H), 7.22-7.16 (m,2H), 6.81 (d, J=7.6 Hz, 1H), 3.75-3.67^(†) (m, 0.38H), 3.46-3.35 (m,1H), 3.25-3.14* (m, 0.48H), 3.07 (s, 4H, 0.33H^(†)), 2.93-2.62 (m, 2H),2.41-2.13 (m, 2H, 0.57H*), 1.98-1.63 (m, 3H), 1.15-1.00 (m, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 169.5*, 168.7^(†), 144.1 (2C), 141.3^(†), 141.0*,164.1 (d, J_(CF)=252.1 Hz, 2C), 138.3 (d, J_(CF)=8.6 Hz, 2C), 133.7(2C), 132.9*, 132.1^(†), 128.1 (2C), 125.8*, 125.5^(†), 124.7 (2C),117.7 (d, J_(CF)=22.0 Hz, 2C), 62.7, 61.5, 56.4, 53.8, 53.7, 52.1, 49.9,49.3, 39.7, 33.8, 29.8, 29.3, 29.1, 23.2, 23.0, 14.0. * and ^(†) labelthe rotamers. IR (thin film): 2970, 2875, 2797, 1644, 1634, 1608, 1591,1548, 1520, 1489, 1455, 1403, 1338, 1290, 1226, 1157, 1108, 1092, 1050,1014, 836 cm⁻¹. MS (ES-API) m/z: 418.2 (100%, [M+H]⁺, C₂₁H₂₅FN₃O₃Srequires 418.2).

4-((4-hydroxyphenyl)thio)-3-nitrobenzoic acid (DS-2-073)

In a 250 mL flask equipped with a reflux condenser,4-fluoro-3-nitrobenzoic acid (9.26 g, 50.0 mmol, 1 eq) and sodiumbicarbonate (4.41 g, 52.5 mmol, 1.05 eq) were suspended in water (100mL). After gas evolution ended, 4-mercaptophenol (6.62 g, 52.5 mmol,1.05 eq) was introduced over 5 min and the orange reaction mixture wasstirred at 90° C. After 4 h, the resulting yellow solid was filtered atroom temperature and washed with water (200 mL) and dried under highvacuum to afford the product (11.97 g, 41.09 mmol, 82%) as a yellowpowder. ¹H NMR (400 MHz, DMSO-d₆) δ 13.51 (s, 1H), 10.21 (s, 1H), 8.64(d, J=1.7 Hz, 1H), 8.02 (dd, J=8.6, 1.8 Hz, 1H), 7.44 (d, J=8.5 Hz, 2H),7.01-6.88 (m, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 165.4, 159.9, 145.1,143.7, 137.7, 133.9, 127.9, 127.8, 126.5, 117.6, 117.1.

N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-4-fluoro-3-nitrobenzamidehydrochloride (DS-2-077)

Following the general procedure B-3 using 4-fluoro-3-nitrobenzoic acidand N-ethyl-N-(prop-2-yn-1-yl)ethane-1,2-diamine (see DS-2-035). Beigesolid (84%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.39 (s, 1H), 9.40 (t, J=5.4Hz, 1H), 8.68 (dd, J=7.3, 2.2 Hz, 1H), 8.40 (ddd, J=8.7, 4.2, 2.3 Hz,1H), 7.73 (dd, J=11.1, 8.8 Hz, 1H), 4.30-4.18 (m, 2H), 3.82 (s, 1H),3.72 (q, J=5.8 Hz, 2H), 3.41-3.15 (m, 4H), 1.28 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, DMSO-d₆) δ 163.7, 156.3 (d, J_(CF)=266.1 Hz), 136.6 (d,J_(CF)=7.7 Hz), 135.3 (d, J_(CF)=10.0 Hz), 130.8 (d, J_(CF)=3.7 Hz),125.5 (d, J_(CF)=1.9 Hz), 118.8 (d, J_(CF)=21.3 Hz), 81.4, 72.7, 50.7,47.9, 40.9, 34.5, 8.8. IR (thin film): 3204, 2948, 2480, 2125, 1661,1652, 1621, 1538, 1495, 1470, 1456, 1352, 1316, 1270 cm⁻¹. MS (ES-API)m/z: 294.1 (100%, [M−Cl]⁺, C₁₄H₁₇FN₃O₃ requires 294.1).

3-nitro-4-((4-(prop-2-yn-1-yloxy)phenyl)thio)benzoic acid (DS-2-079)

A 100 mL flask was charged with the phenol DS-2-073 (2.91 g, 10.0 mmol,1 eq) and a solution of sodium hydroxide (1.20 g, 30.0 mmol, 3 eq) inethanol (50 mL). To the resulting deep purple solution was addedpropargyl bromide (80% wt. in toluene, 3.35 mL, 30.0 mmol, 3 eq) in oneportion and the reaction mixture was heated under reflux. After 3.5 h,the yellow precipitate was filtered at room temperature and washed withethanol (3×50 mL) and dried under high vacuum to afford the product(1.33 g, 4.05 mmol, 41%) as a yellow powder. ¹H NMR (400 MHz, DMSO-d₆) δ8.59 (d, J=1.6 Hz, 1H), 7.92 (dd, J=8.3, 1.7 Hz, 1H), 7.60-7.54 (m, 2H),7.18-7.13 (m, 2H), 6.73 (d, J=8.3 Hz, 1H), 4.90 (d, J=2.4 Hz, 2H), 3.65(t, J=2.3 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆) δ 167.3, 158.9, 143.8,139.2, 138.0, 137.4, 134.4, 126.8, 125.9, 121.3, 116.8, 78.9, 78.8,55.8. IR (thin film): 1586, 1538, 1404, 1327, 1179 cm⁻¹. MS (ES-API)m/z: 328.0 (100%, [M−H]⁻, C₁₆H₁₀NO₅S requires 328.0).

4-(4-azidophenyl)thio)-N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-3-nitrobenzamide(DS-2-091)

A 15 mL pressure tube was charged with the aryl fluoride DS-2-077 (330mg, 1.00 mmol, 1 eq), water (5 mL), 4-aminothiophenol (138 mg, 1.10mmol, 1.1 eq) and potassium carbonate (290 mg, 2.10 mmol, 2.1 eq). Thetube was sealed and the reaction mixture was stirred at 90° C. for 2 hand then overnight at room temperature. The reaction mixture was dilutedwith 1M sodium hydroxide (15 mL) and dichloromethane (20 mL). Theorganic phase was washed with 1M sodium hydroxide (2×20 mL), water (20mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to afford the crude product as an orange thick oil.Purification by column chromatography on silica gel (0-10% MeOH in DCM,MeOH containing 1% NH₃) yielded the aniline (300 mg, 0.75 mmol, 75%) asan orange oil.

A 10 mL flask was charged with the aniline (300 mg, 0.75 mmol, 1 eq) and2M hydrochloric acid (3 mL) and cooled to 0° C. A solution of sodiumnitrite (62 mg, 0.90 mmol, 1.2 eq) in water (0.3 mL) was added dropwise.After 1 h, a solution of sodium azide (73 mg, 1.10 mmol, 1.5 eq) inwater (0.6 mL) was introduced dropwise resulting in rapid gas evolution.The reaction mixture was slowly warmed to room temperature overnight anddiluted with water (15 mL) and dichloromethane (20 mL) and slowlybasified by adding 1M sodium hydroxide (9 mL). The aqueous phase wasextracted with dichloromethane (3×10 mL). The combined organic layerswere washed with water (20 mL) and brine (20 mL), dried over anhydrousmagnesium sulfate and concentrated under reduced pressure to afford thecrude product as yellow solid. Purification by column chromatography onsilica gel (0-2.5% MeOH in DCM, MeOH containing 1% NH₃) yielded theproduct (210 mg, 0.50 mmol, 66%) as yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.61 (d, J=1.6 Hz, 1H), 7.78 (dd, J=8.5, 1.7 Hz, 1H), 7.57 (d,J=8.5 Hz, 2H), 7.16 (d, J=8.5 Hz, 2H), 6.91 (s, 1H), 6.88 (d, J=8.6 Hz,1H), 3.52 (q, J=5.3 Hz, 2H), 3.43 (d, J=2.1 Hz, 2H), 2.78 (t, J=5.8 Hz,2H), 2.62 (q, J=7.1 Hz, 2H), 2.24 (t, J=2.1 Hz, 1H), 1.08 (t, J=7.1 Hz,3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.5, 144.2, 143.3, 142.7, 137.5,131.7, 131.6, 128.2, 125.9, 124.2, 120.9, 78.3, 73.3, 51.4, 47.4, 41.4,37.2, 12.7. IR (thin film): 2971, 2130, 2096, 1637, 1608, 1589, 1548,1520, 1488, 1464, 1338, 1293, 1180, 1106, 1049, 832 cm⁻¹. MS (ES-API)m/z: 425.1 (100%, [M+H]⁺, C₂₀H₂₁N₆O₃S requires 425.1).

N-(2-((4-benzoylbenzyl)(ethyl)amino)ethyl)-3-nitro-4-((4-(prop-2-yn-1-yloxy)phenyl)thio)benzamide(DS-2-093)

A 10 mL flask was charged with the amine DS-2-111 (297 mg, 0.74 mmol, 1eq), acetonitrile (4 mL), potassium carbonate (103 mg, 0.74 mmol, 1 eq)and 4-(bromomethyl)benzophenone (205 mg, 0.74 mmol, 1 eq). The resultingsuspension was stirred vigorously for 16 h. The solid was filtered andthe filtrate concentrated under reduced pressure to afford the crudeproduct as a yellow solid. Purification by column chromatography onsilica gel (0-1% MeOH in DCM, MeOH containing 1% NH₃) yielded theproduct (249 mg, 0.42 mmol, 56%) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.58 (d, J=1.9 Hz, 1H), 7.76-7.69 (m, 5H), 7.59 (tt, J=7.0,7.0, 1.3 Hz, 1H), 7.52-7.41 (m, 6H), 7.12-7.07 (m, 2H), 6.88 (d, J=8.6Hz, 1H), 6.84 (bs, 1H), 4.77 (d, J=2.4 Hz, 2H), 3.70 (s, 2H), 3.49 (q,J=5.4 Hz, 2H), 2.72 (t, J=5.7 Hz, 2H), 2.66 (q, J=7.0 Hz, 2H), 2.59 (t,J=2.4 Hz, 1H), 1.13 (t, J=7.0 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 196.2,164.4, 159.3, 144.4, 144.0, 137.6, 137.5, 136.6, 132.4, 131.4, 131.0,130.3, 129.9, 128.6, 128.3, 128.2, 123.9, 121.5, 116.8, 77.8, 76.2,57.8, 55.9, 51.6, 47.7, 37.3, 11.8. IR (thin film): 3063, 2970, 2934,2818, 2122, 1660, 1652, 1645, 1607, 1548, 1520, 1494, 1463, 1338, 1282,1242, 1176, 1109, 1049, 1023, 925, 831 cm⁻¹. MS (ES-API) m/z: 594.2(100%, [M+H]⁺, C₃₄H₃₂N₃O₅S requires 594.2).

4-(4-benzoylphenoxy)-N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-3-nitrobenzamide(DS-2-097)

Following the general procedure A-4 using the aryl fluoride DS-2-077 and4-hydroxybenzophenone. Purification by column chromatography on silicagel (0-5% MeOH in DCM, MeOH containing 1% NH₃). Yellow oil (88%). ¹H NMR(400 MHz, CDCl₃) δ 8.43 (d, J=2.1 Hz, 1H), 8.05 (dd, J=8.6, 2.2 Hz, 1H),7.91-7.86 (m, 2H), 7.82-7.77 (m, 2H), 7.63-7.58 (m, 1H), 7.50 (t, J=7.6Hz, 2H), 7.18 (d, J=8.6 Hz, 1H), 7.15-7.11 (m, 2H), 7.00 (bs, 1H), 3.56(q, J=5.1 Hz, 2H), 3.48 (d, J=2.3 Hz, 2H), 2.86-2.80 (m, 2H), 2.67 (q,J=7.1 Hz, 2H), 2.27 (t, J=2.3 Hz, 1H), 1.12 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 195.4, 164.4, 159.0, 151.7, 141.3, 137.6, 134.2,133.2, 132.8, 132.7, 130.9, 130.0, 128.5, 125.0, 121.5, 118.5, 78.1,73.8, 51.7, 47.7, 41.5, 37.3, 12.7. IR (thin film): 3066, 2972, 2831,1660, 1652, 1622, 1596, 1537, 1487, 1352, 1307, 1259, 1204, 1167, 1150,1082, 925 cm⁻¹. MS (ES-API) m/z: 472.2 (100%, [M+H]⁺, C₂₇H₂₆N₃O₅requires 472.2).

Ethyl 4-(4-fluorobenzoyl)-3-nitrobenzoate (DS-2-099)

An oven-dried 25 mL flask was charged with ethyl 4-iodo-3-nitrobenzoate(3.21 g, 10.0 mmol, 1 eq) and evacuated and backfilled with argon threetimes. Dry tetrahydrofuran (25 mL) was introduced and the flask wascooled to −40° C. Phenylmagnesium chloride (2M in tetrahydrofuran, 5.5mL, 11 mmol, 1.1 eq) was added dropwise over 1 h and the homogeneoussolution progressively turned from yellow to dark grey. After 10 min,4-fluorobenzaldehyde (1.3 mL, 12.0 mmol, 1.2 eq) was introduced dropwiseover 30 min and the reaction mixture gradually turned dark red. After 30min, the cooling bath was removed and the solution was stirred at roomtemperature for 6 h. Saturated ammonium chloride (10 mL) was added andthe reaction mixture was then poured into water (100 mL). The aqueousphase was extracted with ethyl acetate (3×30 mL) and the combinedorganic layers were washed with brine (100 mL), dried over anhydrousmagnesium sulfate and concentrated under reduced pressure to afford thecrude product as a brown oil. Purification by column chromatography onsilica gel (0-25% ethyl acetate in hexanes) yielded the product as anorange oil (3.02 g) that was used in the next step without furtherpurification.

A 100 mL flask was charged with the alcohol (1.51 g), manganese dioxide(8.69 g, 100 mmol, 20 eq) and dry dichloromethane (50 mL). The resultingblack suspension was stirred at room temperature. After 40 h, furthermanganese dioxide (4.35 g, 50 mmol, 10 eq) was added and the reactionmixture was heated under reflux. After 4 h, the reaction mixture wasfiltered at room temperature through Celite, washing withdichloromethane until the filtrate was colorless. The filtrate wasconcentrated under reduced pressure to afford the crude product as ayellow solid. Purification by column chromatography on silica gel (0-10%EtOAc in Hexanes) yielded the product (869 mg, 2.74 mmol, 55% in twosteps) as a white powder. ¹H NMR (400 MHz, CDCl₃) δ 8.87 (d, J=1.3 Hz,1H), 8.44 (dd, J=7.9, 1.4 Hz, 1H), 7.80-7.44 (m, 2H), 7.57 (d, J=7.8 Hz,1H), 7.18-7.11 (m, 2H), 4.49 (q, J=7.1 Hz, 2H), 1.46 (t, J=7.1 Hz, 3H).¹³C NMR (100 MHz, CDCl₃) δ 191.1, 166.3 (d, J_(CF)=257.1 Hz), 163.8,146.6, 139.4, 134.9, 133.2, 132.0, 131.9, 129.0, 125.7, 116.2 (d,J_(CF)=22.2 Hz), 62.3, 14.3. IR (thin film): 1726, 1678, 1598, 1537,1349, 1284, 1240, 1151, 1112, 1016, 940, 852 cm⁻¹. MS (ES-API) m/z:318.1 (100%, [M+H]⁺, C₁₆H₁₃FNO₅ requires 318.1).

4-(2-benzoylphenoxy)-N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-3-nitrobenzamide(DS-2-101)

Following the general procedure A-4 using the aryl fluoride DS-2-077 and2-hydroxybenzophenone. Purification by column chromatography on silicagel (0-2% MeOH in DCM, MeOH containing 1% NH₃). Yellow oil (34%). ¹H NMR(400 MHz, CDCl₃) δ 8.25 (d, J=2.1 Hz, 1H), 7.88 (dd, J=8.7, 2.2 Hz, 1H),7.75-7.70 (m, 2H), 7.63-7.54 (m, 3H), 7.41 (t, J=7.5 Hz, 3H), 7.16-7.13(m, 1H), 6.93 (d, J=8.7 Hz, 1H), 6.86 (bs, 1H), 3.51 (q, J=5.2 Hz, 2H),3.43 (d, J=2.3 Hz, 2H), 2.79-2.75 (m, 2H), 2.62 (q, J=7.2 Hz, 2H), 2.23(t, J=2.3 Hz, 1H), 1.08 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ194.5, 164.4, 153.1, 152.0, 139.5, 136.7, 133.6, 132.73, 132.69, 132.0,130.9, 129.8, 129.3, 128.4, 125.8, 124.6, 121.0, 119.0, 78.3, 73.3,51.4, 47.4, 41.4, 37.2, 12.7. IR (thin film): 3063, 2972, 2937, 2829,1660, 1652, 1644, 1620, 1602, 1532, 1479, 1449, 1352, 1317, 1294, 1264,1200, 1183, 1149, 1104, 1079, 930 cm⁻¹. MS (ES-API) m/z: 472.2 (100%,[M+H]⁺, C₂₇H₂₅N₃O₅ requires 472.2).

4-(4-fluorobenzoyl)-3-nitrobenzoic acid (DS-2-103)

A 50 mL flask was charged with the ethyl ester (635 mg, 2.00 mmol, 1eq), lithium hydroxide monohydrate (168 mg, 4 mmol, 2 eq) and water (10mL). The resulting suspension was heated under reflux for 2 h. To theresulting homogeneous orange solution was added dropwise 1M hydrochloricacid (4 mL), precipitating a white solid. The reaction mixture wascooled to room temperature and filtered, washing with water (50 mL) toafford the crude product as a white power. Purification by columnchromatography on silica gel (0-60% EtOAc in Hex, 1% AcOH) yielded theproduct (372 mg, 1.29 mmol, 64%) as a white solid. ¹H NMR (400 MHz,DMSO-d₆) δ 13.94 (s, 1H), 8.66 (s, 1H), 8.41 (dd, J=7.8, 1.2 Hz, 1H),7.87-7.83 (m, 2H), 7.81 (d, J=7.9 Hz, 1H), 7.42-7.36 (m, 2H). ¹³C NMR(100 MHz, DMSO-d₆) δ 191.3, 165.6 (d, J_(CF)=253.9 Hz), 165.0, 146.2,138.3, 135.2, 133.6, 132.3 (d, J_(CF)=9.8 Hz), 132.0, 129.6, 125.4,116.3 (d, J_(CF)=22.3 Hz). IR (thin film): 1704, 1681, 1596, 1532, 1505,1417, 1351, 1281, 1243, 1150, 857 cm⁻¹. MS (ES-API) m/z: 288.0 (100%,[M−H]⁻, C₁₄H₇FNO₅ requires 288.0), 577.0 (20%, [2M−H]⁻).

N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-4-(4-fluorobenzoyl)-3-nitrobenzamide(DS-2-107)

A 10 mL oven-dried flask equipped with a reflux condenser was chargedwith the carboxylic acid (145 mg, 0.50 mmol, 1 eq) and thionyl chloride(1.5 mL) and heated under reflux 4 h. The resulting homogeneous mixturewas then concentrated to dryness under reduced pressure. The white solidobtained was then dissolved in dry dichloromethane (2 mL) and thesolvent was evaporated. This process was repeated twice yielding theacyl chloride as a white solid.

A 10 mL oven-dried flask was charged with the acyl chloride, drydichloromethane (1.5 mL) and 4-dimethylaminopyridine (1 mg, 0.008 mmol,1 mol %) and cooled to 0° C. A solution ofN-ethyl-N-(prop-2-yn-1-yl)ethane-1,2-diamine (see DS-2-035) (95 mg, 0.75mmol, 1.5 eq) in dry dichloromethane (1.5 mL) was added dropwise over 10min and the reaction mixture was slowly warmed to room temperature.After 16 h, the reaction mixture was diluted with dichloromethane (20mL) and water (20 mL) and basified with 1M sodium hydroxide to reachpH˜12. The aqueous phase was extracted with dichloromethane (3×10 mL).The combined organic layers were washed with water (30 mL), brine (30mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to afford the crude product as a yellow oil.Purification by column chromatography on silica gel (0-5% MeOH in DCM,MeOH containing 1% NH₃) yielded the product (169 mg, 0.43 mmol, 85%) asa light yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 8.62 (d, J=1.4 Hz, 1H),8.21 (dd, J=7.8, 1.5 Hz, 1H), 7.82-7.72 (m, 2H), 7.55 (d, J=7.8 Hz, 1H),7.17-7.11 (m, 2H), 7.08 (bs, 1H), 3.59 (q, J=5.2 Hz, 2H), 3.47 (d, J=2.3Hz, 2H), 2.86-2.81 (m, 2H), 2.65 (q, J=7.1 Hz, 2H), 2.27 (t, J=2.3 Hz,1H), 1.11 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.2, 166.3 (d,J_(CF)=257.1 Hz), 164.1, 146.6, 138.0, 137.3, 132.7, 132.0 (d,J_(CF)=3.1 Hz), 131.9 (d, J_(CF)=9.6 Hz), 129.2, 123.1, 116.2 (d,J_(CF)=22.2 Hz), 78.3, 73.4, 51.4, 47.5, 41.4, 37.4, 12.7. IR (thinfilm): 3078, 2973, 2939, 2831, 2361, 1674, 1652, 1645, 1598, 1538, 1506,1349, 1307, 1281, 1241, 1184, 1152, 940, 852 cm⁻¹. MS (ES-API) m/z:398.2 (100%, [M+H]⁺, C₂₁H₂₁FN₃O₄ requires 398.1).

N-(2-(ethylamino)ethyl)-3-nitro-4-((4-(prop-2-yn-1-yloxy)phenyl)thio)benzamide(DS-2-111)

A 10 mL oven-dried flask equipped with a reflux condenser, fitted with adrying tube, was charged with the acid carboxylic DS-2-079 (1.15 g, 3.50mmol, 1 eq) and thionyl chloride (3.5 mL) and heated to reflux. After 4h, the resulting homogeneous mixture was concentrated to dryness underreduced pressure. The solid residue obtained was then dissolved in drydichloromethane (3 mL) and the solvent was evaporated. This process wasrepeated twice yielding the acyl chloride as a yellow-greenish solid.

Dry dichloromethane (2.3 mL) and 4-dimethylaminopyridine (4 mg, 0.04mmol, 1 mol %) were introduced and the flask was cooled to 0° C. Asolution of N-ethyl ethylenediamine (0.37 mL, 3.50 mmol, 1 eq) in drydichloromethane (1.2 mL) was added dropwise over 15 min and theresulting yellow solution was slowly warmed to room temperature. After 3h, the reaction mixture was concentrated to dryness. Purification bycolumn chromatography on silica gel (0-10% MeOH in DCM, MeOH containing1% NH₃) afforded the product (297 mg, 0.74 mmol, 21%) as a yellow solid.¹H NMR (400 MHz, CDCl₃) δ 8.63 (d, J=1.8 Hz, 1H), 7.79 (dd, J=8.6, 1.9Hz, 1H), 7.52-7.47 (m, 2H), 7.24 (s, 1H), 7.11-7.07 (m, 2H), 6.86 (d,J=8.6 Hz, 1H), 4.76 (d, J=2.4 Hz, 2H), 3.53 (q, J=5.4 Hz, 2H), 2.91-2.84(m, 2H), 2.69 (q, J=7.1 Hz, 2H), 2.58 (t, J=2.4 Hz, 1H), 2.29 (bs, 1H),1.12 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 159.4, 144.3,144.1, 137.7, 131.8, 131.3, 128.2, 124.4, 121.7, 116.9, 77.9, 76.4,56.1, 48.1, 43.8, 39.6, 15.2. IR (thin film): 3293, 2969, 1652, 1644,1607, 1591, 1574, 1548, 1520, 1494, 1463, 1338, 1290, 1242, 1176, 1109,1049, 1023, 926, 831 cm⁻¹. MS (ES-API) m/z: 400.1 (100%, [M+H]⁺,C₂₀H₂₂N₃O₄S requires 400.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-((4-fluorophenyl)thio)-4-nitrobenzamide(DS-2-115)

Following the general procedure B-3 using DS-2-113 and2-(aminomethyl)-1-ethylpyrrolidine. The resulting suspension was dilutedwith dichloromethane (10 mL), washed with saturated sodium bicarbonate(2×5 mL), dried over anhydrous magnesium sulfate and concentrated underreduced pressure to afford the crude product. Purification by columnchromatography on silica gel (0-10% MeOH in DCM, MeOH containing 1%NH₃). Yellow solid (85%). ¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J=8.5 Hz,1H), 7.64 (d, J=8.5 Hz, 1H), 7.56 (dd, J=8.3, 5.4 Hz, 2H), 7.36 (bs,1H), 7.16 (t, J=8.4 Hz, 2H), 3.65-3.54 (m, 1H), 3.32-3.18 (m, 2H),2.86-2.71 (m, 2H), 2.39-2.23 (m, 2H), 1.96-1.82 (m, 1H), 1.79-1.69 (m,1H), 1.65-1.46 (m, 2H), 1.09 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 165.4, 164.1 (d, J_(CF)=252.0 Hz), 146.1, 140.0, 138.9, 138.2 (d,J_(CF)=8.6 Hz), 127.0, 126.3, 125.7 (d, J_(CF)=3.5 Hz), 123.7, 117.7 (d,J_(CF)=22.0 Hz), 62.9, 53.6, 48.7, 40.6, 28.1, 23.2, 13.4. IR (thinfilm): 3064, 2970, 2876, 2801, 1667, 1652, 1590, 1574, 1516, 1490, 1456,1336, 1306, 1224, 1156, 1111, 1014, 837 cm⁻¹. MS (ES-API) m/z: 404.2(100%, [M+H]⁺, C₂₀H₂₃FN₃O₃S requires 404.1).

4-(4-fluorophenyl)thio)-N-(2-(4-methylpiperazin-1-yl)ethyl)-3-nitrobenzamide(DS-2-121)

Following the general procedure B-2 using2-(4-methyl-piperazin-1-yl)-ethylamine. Yellow solid (74%). ¹H NMR (400MHz, CDCl₃) δ 8.64 (s, 1H), 7.82 (d, J=8.0 Hz, 1H), 7.62-7.54 (m, 2H),7.22 (t, J=8.5 Hz, 2H), 7.08 (bs, 1H), 6.88 (d, J=8.5 Hz, 1H), 3.56 (q,J=5.2 Hz, 2H), 2.72-2.49 (m, 10H), 2.34 (s, 3H). ¹³C NMR (100 MHz,CDCl₃) δ 164.6, 164.3 (d, J_(CF)=252.3 Hz), 144.4, 143.5, 138.3 (d,J_(CF)=8.6 Hz), 131.9, 131.6, 128.3, 125.6 (d, J_(CF)=3.5 Hz), 124.4,117.9 (d, J_(CF)=22.0 Hz), 56.3, 54.8, 52.7, 46.0, 36.4. IR (thin film):3284, 2942, 2796, 1634, 1607, 1551, 1520, 1490, 1456, 1334, 1286, 1228,1168, 1106, 1091, 1049, 1012, 841 cm⁻¹. MS (ES-API) m/z: 419.2 (100%,[M+H]⁺, C₂₀H₂₄FN₄O₃S requires 419.2).

4-((4-fluorophenyl)thio)-3-nitro-N-(2-(piperidin-1-yl)ethyl)benzamide(DS-2-125)

Following the general procedure B-2 using 1-(2-aminoethyl)piperidine.Yellow solid (72%). ¹H NMR (400 MHz, CDCl₃) δ 8.72 (s, 1H), 7.89 (d,J=8.0 Hz, 1H), 7.63-7.53 (m, 2H), 7.25-7.17 (m, 2H), 6.88 (d, J=8.5 Hz,1H), 3.65-3.55 (m, 2H), 2.70 (bs, 2H), 2.60 (bs, 4H), 1.71 (bs, 4H),1.52 (bs, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 164.5, 164.2 (d, J_(CF)=252.3Hz), 144.4, 143.2, 138.3 (d, J_(CF)=8.6 Hz), 131.8, 131.7, 128.2, 125.6(d, J_(CF)=3.5 Hz), 124.5, 117.8 (d, J_(CF)=22.0 Hz), 56.7, 54.3, 36.6,26.1, 24.3. IR (thin film): 2940, 2782, 1634, 1608, 1591, 1538, 1520,1506, 1493, 1470, 1332, 1288, 1224, 1159, 1131, 1106, 1048, 834 cm⁻¹. MS(ES-API) m/z: 404.1 (100%, [M+H]⁺, C₂₀H₂₃FN₃O₃S requires 404.1).

tert-butyl4-(2-(4-((4-fluorophenyl)thio)-3-nitrobenzamido)ethyl)piperazine-1-carboxylate(DS-2-127)

Following the general procedure B-2 using4-(2-aminoethyl)-1-boc-piperazine. Yellow solid (79%). ¹H NMR (400 MHz,CDCl₃) δ 8.70 (s, 1H), 7.86 (s, 1H), 7.63-7.53 (m, 2H), 7.25-7.18 (m,2H), 6.88 (d, J=8.5 Hz, 1H), 3.71-3.45 (m, 6H), 2.83-2.47 (m, 6H), 1.46(s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 164.6, 164.2 (d, J_(CF)=252.4 Hz),154.8, 144.3, 143.4, 138.3 (d, J_(CF)=8.6 Hz), 131.7, 131.6, 128.2,125.4 (d, J_(CF)=3.2 Hz), 124.3, 117.8 (d, J_(CF)=22.0 Hz), 79.9, 56.4,52.7, 43.6 (bs), 36.6, 28.5. IR (thin film): 3070, 2978, 2937, 2815,1694, 1682, 1668, 1652, 1645, 1608, 1590, 1548, 1520, 1462, 1424, 1366,1338, 1293, 1243, 1159, 1131, 1050, 1005, 837 cm⁻¹. MS (ES-API) m/z:505.2 (100%, [M+H]⁺, C₂₄H₃₀FN₄O₅S requires 505.2).

4-((4-fluorophenyl)thio)-3-nitro-N-(2-(piperazin-1-yl)ethyl)benzamide(DS-2-129)

A 10 mL flask was charged with DS-2-127 (505 mg, 1.00 mmol, 1 eq) and asolution of trifluoroacetic acid in dichloromethane (20%, 5 mL) wasintroduced. The reaction mixture was stirred overnight and concentratedto dryness under reduced pressure. The oily residue was dissolved indichloromethane (20 mL) and saturated sodium bicarbonate (20 mL). Theaqueous phase was extracted with dichloromethane (2×10 mL) and thecombined organic layers were washed with brine (30 mL), dried overanhydrous magnesium sulfate and concentrated under reduced pressure toafford the crude product. Purification by column chromatography onsilica gel (0-20% MeOH in DCM, MeOH containing 1% NH₃) afforded theproduct (239 mg, 0.59 mmol, 59%) as a yellow solid. ¹H NMR (400 MHz,CDCl₃) δ 8.63 (d, J=1.8 Hz, 1H), 7.79 (dd, J=8.5, 1.9 Hz, 1H), 7.61-7.55(m, 2H), 7.25-7.18 (m, 2H), 6.95 (bs, 1H), 6.88 (d, J=8.5 Hz, 1H), 3.55(q, J=5.5 Hz, 2H), 2.98-2.93 (m, 4H), 2.62 (t, J=5.9 Hz, 2H), 2.54 (s,4H), 2.40 (bs, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 164.6, 164.2 (d,J_(CF)=252.4 Hz), 144.2, 143.3, 138.3 (d, J_(CF)=8.6 Hz), 131.8, 131.7,128.2, 125.5 (d, J_(CF)=3.4 Hz), 124.4, 117.8 (d, J_(CF)=22.0 Hz), 56.8,53.8, 45.9, 36.4. IR (thin film): 2945, 2821, 1652, 1634, 1608, 1590,1548, 1520, 1490, 1464, 1338, 1226, 1158, 1049, 837 cm⁻¹. MS (ES-API)m/z: 405.1 (100%, [M+H]⁺, C₁₉H₂₂FN₄O₃S requires 405.1).

4-((4-chloro-3-(trifluoromethyl)phenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(DS-2-143)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and4-chloro-3-trifluoromethyl-benzenethiol. Pale yellow solid (78%). ¹H NMR(400 MHz, CDCl₃) δ 8.64 (s, 1H), 7.91 (s, 1H), 7.80 (d, J=8.0 Hz, 1H),7.72-7.62 (m, 2H), 7.04 (bs, 1H), 6.87 (d, J=8.2 Hz, 1H), 3.72-3.60 (m,1H), 3.29 (d, J=13.0 Hz, 1H), 3.18 (s, 1H), 2.85-2.75 (m, 1H), 2.69 (s,1H), 2.31-2.14 (m, 2H), 1.97-1.85 (m, 1H), 1.79-1.63 (m, 2H), 1.63-1.53(m, 1H), 1.10 (t, J=6.5 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.7,144.8, 141.0, 140.1, 135.0, 134.6 (q, J_(CF)=5.2 Hz), 133.5, 132.6,131.8, 130.4 (q, J_(CF)=32.0 Hz), 130.2, 128.3, 124.7, 122.2 (q,J_(CF)=274.0 Hz), 62.1, 53.6, 48.1, 41.1, 28.3, 23.0, 14.1. IR (thinfilm): 3096, 2970, 2801, 1644, 1607, 1522, 1469, 1396, 1338, 1307, 1254,1178, 1144, 1110, 1037 cm⁻¹. MS (ES-API) m/z: 488.1 (100%, [M+H]⁺,C₂₁H₂₂ClF₃N₃O₃S requires 488.1).

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-((3-(trifluoromethyl)phenyl)thio)benzamide(JCH-107)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and3-trifluorobenzenethiol. Light yellow solid (72%). ¹H NMR (400 MHz,CDCl₃) δ 8.68 (s, 1H), 7.87 (s, 1H), 7.83-7.76 (m, 3H), 7.68-7.62 (m,1H), 7.14 (bs, 1H), 6.87 (d, J=8.4 Hz, 1H), 3.70 (ddd, J=13.9, 6.8, 2.7Hz, 1H), 3.36-3.30 (m, 1H), 3.22 (t, J=7.2 Hz, 1H), 2.83 (dq, J=11.8,7.4 Hz, 1H), 2.74 (bs, 1H), 2.34-2.16 (m, 2H), 1.98-1.87 (m, 1H),1.81-1.56 (m, 3H), 1.13 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ164.8, 144.9, 141.8, 139.3, 132.9 (q, J_(CF)=32.9 Hz), 132.6 (q,J_(CF)=3.7 Hz), 132.3, 132.1, 131.8, 130.9, 128.5, 127.3 (q, J_(CF)=3.6Hz), 124.7, 123.5 (q, J_(CF)=271.5), 62.5, 53.7, 48.4, 40.8, 28.2, 23.1,14.0. IR (thin film): 3073, 2971, 2877, 2805, 1644, 1608, 1548, 1523,1338, 1323.5, 1306, 1170, 1130, 1111 cm⁻¹. MS (ES-API) m/z: 454.1 (100%,[M+H]⁺, C₂₁H₂₃F₃N₃O₃S requires 454.1). mp: 136° C.

4-((3-aminophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(JCH-109)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and3-aminothiophenol. The resulting biphasic mixture was diluted withdichloromethane and washed with saturated sodium bicarbonate, water andbrine, dried over anhydrous magnesium sulfate and concentrated underreduced pressure. Purification by column chromatography on silica gel(5-10% MeOH in DCM, MeOH containing 1% NH₃). Orange solid (64%). ¹H NMR(400 MHz, CDCl₃) δ 8.63 (d, J=2.0 Hz, 1H), 7.74 (dd, J=8.5, 1.8 Hz, 1H),7.27-7.22 (m, 1H), 7.12 (bs, 1H), 7.00 (d, J=8.4 Hz, 1H), 6.93 (d, J=7.6Hz, 1H), 6.89-6.86 (m, 1H), 6.80 (dd, J=8.0, 2.0 Hz, 1H), 3.89 (s, 2H),3.68 (ddd, J=13.8, 7.3, 2.9 Hz, 1H), 3.31 (dt, J=14, 3.6 Hz, 1H),3.23-3.17 (m, 1H), 2.82 (dq, J=12.1, 7.4 Hz, 1H), 2.75-2.68 (m, 1H),2.32-2.17 (m, 2H), 1.96-1.86 (m, 1H), 1.77-1.55 (m, 3H), 1.12 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 148.2, 144.4, 143.8, 131.5,131.4, 131.2, 130.8, 128.7, 125.5, 124.5, 121.6, 117.0, 62.5, 53.6,48.3, 40.9, 28.2, 23.1, 14.0. IR (thin film): 3095, 2969, 2876, 2809,1644, 1607, 1594, 1546, 1519, 1482, 1465, 1337, 1294, 1049 cm⁻¹. MS(ES-API) m/z: 401.1 (38%, [M+H]⁺, C₂₀H₂₅N₄O₃S requires 401.2). mp:56-62° C.

4-((2-chlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(JCH-111)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-chlorothiophenol. Purification by column chromatography on silica gel(5-10% MeOH in DCM, MeOH containing 1% NH₃). Yellow solid (62%). ¹H NMR(400 MHz, CDCl₃) δ 8.69 (d, J=1.6 Hz, 1H), 7.80 (dd, J=7.2, 1.2 Hz, 1H),7.72 (d, J=7.6 Hz, 1H), 7.59 (d, J=8 Hz, 1H), 7.51-7.46 (m, 1H),7.42-7.37 (m, 1H), 7.17 (bs, 1H), 6.78 (d, J=8.4 Hz, 1H), 3.69 (ddd,J=13.8, 7.3, 3.0 Hz, 1H), 3.32 (dt, J=14.0, 3.6 Hz, 1H), 3.25-3.18 (m,1H), 2.83 (dq, J=12.1, 7.4 Hz, 1H), 2.77-2.70 (m, 1H), 2.34-2.19 (m,2H), 1.97-1.87 (m, 1H), 1.79-1.56 (m, 3H), 1.12 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.0, 144.6, 141.1, 140.2, 138.3, 132.3, 132.0,131.7, 131.2, 129.4, 128.5, 127.9, 124.8, 62.6, 53.6, 48.4, 40.8, 28.2,23.1, 13.9. IR (thin film): 3063, 2970, 2876, 2805, 1651, 1608, 1548,1520, 1465, 1451, 1338, 1295, 1243, 1036, 748 cm⁻¹. MS (ES-API) m/z:420.1 (100% [M+H]⁺, C₂₀H₂₃ClN₃O₃S requires 420.1). mp: 135-137° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(o-tolylthio)benzamide(JCH-114)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-methylthiophenol. The yellow precipitate was filtered, dissolved indichloromethane, washed with 1M sodium hydroxide (3×), water, and brine.The organic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. Purification by columnchromatography on silica gel (5-10% MeOH in DCM, MeOH containing 1%NH₃). Light yellow solid (31%). ¹H NMR (400 MHz, CDCl₃) δ 8.70 (d, J=1.6Hz, 1H), 7.76 (dd, J=8.6, 2.0 Hz, 1H), 7.57 (d, J=7.2 Hz, 1H), 7.47-7.38(m, 2H), 7.33-7.28 (m, 1H), 7.22 (bs, 1H), 6.74 (d, J=8.4 Hz, 1H), 3.70(ddd, J=13.9, 7.4, 3.1 Hz, 1H), 3.32 (dt, J=13.9, 3.5 Hz, 1H), 3.24-3.18(m, 1H), 2.83 (dq, 12.0, 7.4 Hz, 1H), 2.77-2.70 (m, 1H), 2.34-2.18 (m,2H), 2.33 (s, 3H), 1.96-1.86 (m, 1H), 1.79-1.56 (m, 3H), 1.12 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 144.6, 143.4, 142.6, 137.3,131.64, 131.60, 131.4, 131.2, 129.2, 127.9, 127.6, 124.8, 62.7, 53.6,48.4, 40.8, 28.2, 23.1, 20.6, 13.9. IR (thin film): 3063, 2970, 2876,2804, 1643, 1608, 1547, 1521, 1467, 1338, 1294, 1048, 750 cm⁻¹. MS(ES-API) m/z: 400.2 (100% [M+H]⁺, C₂₁H₂₆N₃O₃S requires 400.2). mp:88-91° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((2-methoxyphenyl)thio)-3-nitrobenzamide(JCH-117)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-methoxythiophenol. The yellow precipitate was filtered, dissolved indichloromethane, washed with 1M sodium hydroxide (3×), water, and brine.The organic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. Purification by columnchromatography on silica gel (5-10% MeOH in DCM, MeOH containing 1%NH₃). Light yellow solid (28%). ¹H NMR (400 MHz, CDCl₃) δ 8.70 (s, 1H),7.80 (d, J=8.4 Hz, 1H), 7.59-7.50 (m, 2H), 7.43 (s, 1H), 7.09-7.01 (m,2H), 6.85 (d, J=8.4 Hz, 1H), 3.77 (s, 3H), 3.75-3.67 (m, 1H), 3.42-3.35(m, 1H), 3.32-3.26 (m, 1H), 2.93-2.82 (m, 2H), 2.43-2.28 (m, 2H),2.00-1.90 (m, 1H), 1.83-1.61 (m, 3H), 1.16 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 165.2, 160.3, 144.6, 142.8, 137.9, 132.9, 131.3,131.1, 128.1, 124.8, 122.0, 117.6, 112.0, 63.3, 56.1, 53.7, 48.9, 40.8,28.1, 23.2, 13.5. IR (thin film): 3272, 3068, 2969, 2803, 1638, 1608,1549, 1521, 1476, 1341, 1295, 1276, 1250, 751 cm⁻¹. MS (ES-API) m/z:416.2 (100%, [M+H]⁺, C₂₁H₂₆N₃O₄S requires 416.2). mp: 104-107° C.

4-(2,4-dichlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(JCH-120)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2,4-dichlorothiophenol. Purification by column chromatography on silicagel (5-10% MeOH in DCM, MeOH containing 1% NH₃). Light yellow solid(44%). ¹H NMR (400 MHz, CDCl₃) δ 8.67 (d, J=2.0 Hz, 1H), 7.80 (dd,J=8.5, 2.0 Hz, 1H), 7.65 (d, J=8.3 Hz, 1H), 7.61 (d, J=2.2 Hz, 1H), 7.39(dd, J=8.2, 2.2 Hz, 1H), 7.06 (bs, 1H), 6.78 (d, J=8.4 Hz, 1H), 3.68(ddd, J=13.8, 7.3, 2.7 Hz, 1H), 3.33-3.27 (m, 1H), 3.22-3.16 (m, 1H),2.81 (dq, J=12.0, 7.4 Hz, 1H), 2.73-2.66 (m, 1H), 2.31-2.16 (m, 2H),1.96-1.86 (m, 1H), 1.79-1.54 (m, 3H), 1.11 (t, J=7.2 Hz, 3H). ¹³C NMR(100 MHz, CDCl₃) δ 164.9, 144.7, 141.1, 140.4, 138.9, 138.0, 132.4,131.8, 131.1, 128.9, 128.1, 127.8, 124.8, 62.2, 53.6, 48.2, 40.9, 28.3,23.1, 14.1. IR (thin film): 3081, 2970, 2938, 2876, 2804, 1644, 1607,1548, 1520, 1454, 1338, 1294, 1243, 1098, 1049, 815, 737 cm⁻¹. MS(ES-API) m/z: 454.1 (100%, [M+H]⁺, C₂₀H₂₂Cl₂N₃O₃S requires 454.1). mp:135-137° C.

4-((3,5-dichlorophenyl)thio)-N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(JCH-124)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and3,5-dichlorothiophenol. Purification by column chromatography on silicagel (5-10% MeOH in DCM, MeOH containing 1% NH₃). Light yellow solid(61%). ¹H NMR (400 MHz, CDCl₃) δ 8.67 (d, J=2.0 Hz, 1H), 7.85 (dd,J=8.5, 2.0 Hz, 1H), 7.51-7.49 (m, 1H), 7.48-7.46 (m, 2H), 7.21 (bs, 1H),6.95 (d, J=8.5 Hz, 1H), 3.70 (ddd, J=13.8, 7.4, 3.0 Hz, 1H), 3.35-3.29(m, 1H), 3.23-3.17 (m, 1H), 2.88-2.78 (m, 1H), 2.76-2.70 (m, 1H),2.33-2.19 (m, 2H), 1.97-1.87 (m, 1H), 1.79-1.56 (m, 3H), 1.12 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 144.9, 141.0, 136.6, 133.9,133.7, 132.6, 131.9, 130.8, 128.7, 124.7, 62.5, 53.6, 48.3, 40.8, 28.1,23.1, 14.0. IR (thin film): 3072, 2970, 2938, 2805, 1644, 1608, 1559,1522, 1466, 1406, 1339, 1294, 1141, 1106, 1048, 800, 736 cm⁻¹. MS(ES-API) m/z: 454.1 (100%, [M+H]⁺, C₂₀H₂₂Cl₂N₃O₃S requires 454.1). mp:44-47° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-3-nitro-4-(quinolin-2-ylthio)benzamide(JCH-127)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-quinolinethiol. The reaction mixture was diluted with dichloromethane,washed with 1M sodium hydroxide (3×), water, and brine. The organiclayer was dried over anhydrous magnesium sulfate and concentrated underreduced pressure. Purification by column chromatography on silica gel(0-10% MeOH in DCM, MeOH containing 1% NH₃). Yellow solid (41%). ¹H NMR(400 MHz, CDCl₃) δ 8.60 (s, 1H), 8.16 (d, J=8.4 Hz, 1H), 8.01 (d, J=8.5Hz, 1H), 7.89-7.82 (m, 2H), 7.77-7.72 (m, 1H), 7.59 (t, J=8 Hz, 1H),7.52-7.47 (m, 2H), 7.18 (bs, 1H), 3.75-3.67 (m, 1H), 3.38-3.31 (m, 1H),3.22 (t, J=7.2 Hz, 1H), 2.89-2.79 (m, 1H), 2.78-2.70 (m, 1H), 2.34-2.17(m, 3H), 1.99-1.88 (m, 1H), 1.82-1.58 (m, 3H), 1.31 (t, J=6.8 Hz, 3H).¹³C NMR (100 MHz, CDCl₃) δ 164.9, 155.1, 148.7, 147.5, 137.8, 136.6,133.7, 132.1, 131.2, 130.6, 129.1, 127.8, 127.5, 127.2, 124.2 (2C),62.4, 53.6, 48.3, 40.9, 28.1, 23.0, 14.0. IR (thin film): 3067, 2970,2876, 2807, 1652, 1608, 1589, 1524, 1467, 1422, 1340, 1295, 1138, 1097,910, 733 cm⁻¹. MS (ES-API) m/z: 437.2 (58%, [M+H]⁺, C₂₃H₂₅N₄O₃S requires437.2). mp: 40-43° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((3-hydroxyphenyl)thio)-3-nitrobenzamide(JCH-140)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and3-mercaptophenol. The reaction mixture was diluted with dichloromethane(20 mL) and methanol (5 mL), washed with saturated sodium bicarbonate(3×10 mL), water (10 mL), and brine (10 mL). The organic layer was driedover anhydrous magnesium sulfate and concentrated under reducedpressure. Purification by column chromatography on silica gel (5-10%MeOH in DCM, MeOH containing 1% NH₃). Orange solid (14%). ¹H NMR (400MHz, CDCl₃) δ 8.66 (d, J=1.6 Hz, 1H), 7.73 (dd, J=8.6, 1.9 Hz, 1H), 7.60(bs, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 7.03-7.00 (m,1H), 6.98-6.94 (m, 1H), 6.89 (d, J=8.8 Hz, 1H), 4.26 (bs, 1H), 3.79-3.71(m, 1H), 3.43 (dd, J=14.1, 3.8 Hz, 1H), 3.32-3.25 (m, 1H), 2.96-2.84 (m,2H), 2.45-2.30 (m, 2H), 2.02-1.92 (m, 1H), 1.85-1.62 (m, 3H), 1.15 (t,J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.7, 158.7, 144.3, 144.0,131.5, 131.4, 130.9, 130.7, 128.5, 126.9, 124.6, 123.1, 118.7, 63.5,53.6, 48.9, 40.9, 27.9, 23.0, 13.4. IR (thin film): 3066, 2972, 2811,1652, 1645, 1607, 1548, 1520, 1464, 1456, 1338, 1301, 1262, 1242, 1049,736 cm⁻¹. MS (ES-API) m/z: 402.2 (100% [M+H]⁺, C₂₀H₂₄N₃O₄S requires402.1). mp: 77-80° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((2-hydroxyphenyl)thio)-3-nitrobenzamide(JCH-143)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-mercaptophenol. The reaction mixture was diluted with dichloromethane(20 mL) and methanol (5 mL), washed with saturated sodium bicarbonate(3×10 mL), water (10 mL), and brine (10 mL). The organic layer was driedover anhydrous magnesium sulfate and concentrated under reducedpressure. Purification by column chromatography on silica gel (5-10%MeOH in DCM, MeOH containing 1% NH₃). Orange solid (54%). ¹H NMR (400MHz, CDCl₃) δ 8.60 (s, 1H), 7.88 (bs, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.47(d, J=7.6 Hz, 1H), 7.40 (t, J=7.6 Hz, 1H), 7.04 (d, J=8.2 Hz, 1H), 6.94(t, J=7.5 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 4.41 (bs, 1H), 3.80-3.70 (m,1H), 3.45-3.37 (m, 1H), 3.31-3.24 (m, 1H), 2.98-2.83 (m, 2H), 2.45-2.31(m, 2H), 1.97-1.85 (m, 1H), 1.82-1.68 (m, 2H), 1.67-1.57 (m, 1H), 1.09(t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 161.6, 144.5,142.1, 137.5, 133.4, 131.01, 130.99, 127.7, 125.4, 119.9, 118.8, 115.5,64.7, 53.4, 49.0, 40.1, 27.4, 22.9, 12.7. IR (thin film): 3286, 3060,2971, 2877, 2806, 1643, 1606, 1544, 1520, 1454, 1387, 1338, 1290, 1255,1050, 844, 753, 736 cm⁻¹. MS (ES-API) m/z: 402.2 (100%, [M+H]⁺,C₂₀H₂₄N₃O₄S requires 402.1). mp: 109-113° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((3-methoxyphenyl)thio)-3-nitrobenzamide(JCH-146)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and3-methoxybenzenethiol. Orange solid (67%). ¹H NMR (400 MHz, CDCl₃) δ8.65 (d, J=1.2 Hz, 1H), 7.77 (dd, J=8.4, 1.2 Hz, 1H), 7.41 (t, J=7.9 Hz,1H), 7.17 (d, J=7.2 Hz, 1H), 7.13-7.03 (m, 2H), 6.96 (d, 8.8 Hz, 1H),3.83 (s, 3H), 3.69 (ddd, J=13.8, 7.3, 3.0 Hz, 1H), 3.36-3.28 (m, 1H),3.22 (t, J=6.8 Hz, 1H), 2.83 (dq, J=12.0, 7.4 Hz, 1H), 2.73 (bs, 1H),2.34-2.18 (m, 3H), 1.98-1.86 (m, 1H), 1.80-1.56 (m, 3H), 1.13 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 165.1, 160.8, 144.4, 143.3, 131.7,131.5, 131.24, 131.21, 128.5, 128.1, 124.5, 120.8, 116.6, 62.4, 55.6,53.6, 48.3, 40.9, 28.2, 23.1, 14.0. IR (thin film): 3069, 2969, 2876,2806, 1643, 1608, 1590, 1548, 1521, 1467, 1338, 1285, 1248, 1040, 748,736 cm⁻¹. MS (ES-API) m/z: 416.2 (100%, [M+H]⁺, C₂₁H₂₆N₃O₄S requires416.2). mp: 103-105° C.

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((2-(hydroxymethyl)phenyl)thio)-3-nitrobenzamide(JCH-149)

Following the general procedure A-3 using the aryl fluoride DS-1-153 and2-mercaptobenzyl alcohol. The reaction mixture was diluted withdichloromethane (20 mL) and methanol (5 mL), washed with saturatedsodium bicarbonate (3×10 mL), water (10 mL), and brine (10 mL). Theorganic layer was dried over anhydrous magnesium sulfate andconcentrated under reduced pressure. Purification by columnchromatography on silica gel (0-5% MeOH in DCM, MeOH containing 1% NH₃).Yellow solid (40%). ¹H NMR (400 MHz, CDCl₃) δ 8.72 (s, 1H), 7.77 (d,J=8.5 Hz, 1H), 7.70 (d, J=7.9 Hz, 1H), 7.60-7.55 (m, 2H), 7.46-7.31 (m,2H), 6.73 (dd, J=8.8, 0.8 Hz, 1H), 4.75 (s, 2H), 3.76-3.69 (m, 1H),3.40-3.32 (m, 1H), 3.29-3.21 (m, 1H), 2.92-2.77 (m, 2H), 2.60 (bs, 1H),2.39-2.23 (m, 2H), 1.98-1.88 (m, 1H), 1.80-1.59 (m, 3H), 1.15 (t, J=6.8Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 145.5, 144.5, 142.1, 137.3,131.52, 131.48, 131.3, 129.2, 128.8, 127.7, 127.6, 124.9, 62.8, 62.5,53.5, 48.4, 40.5, 27.6, 22.8, 13.7. IR (thin film): 3068, 2972, 2877,2814, 1648, 1608, 1547, 1522, 1466, 1339, 1295, 1047, 910, 733 cm⁻¹. MS(ES-API) m/z: 416.2 (100%, [M+H]⁺, C₂₁H₂₆N₃O₄S requires 416.2). mp:59-63° C.

tert-butyl2-((4-((4-fluorophenyl)thio)-3-nitrobenzamido)methyl)pyrrolidine-1-carboxylate(WW3-79)

A mixture of 4-((4-fluorophenyl)thio)-3-nitrobenzoic acid (0.7478 g,2.55 mmol), DMAP (0.0669 g, 0.55 mmol), EDC hydrochloride (0.5371 g,2.80 mmol) and CH₂Cl₂ (12 mL) was stirred at room temperature for 1 h.tert-Butyl 2-(aminomethyl)pyrrolidine-1-carboxylate (0.5012 g, 2.51mmol) in CH₂Cl₂ (2 mL) was then added and the resulting solution wasstirred at room temperature for 21 h. The reaction solution was dilutedwith CH₂Cl₂ (30 mL), and was washed sequentially with saturated aq. NaCl(30 mL) and saturated aq. NaHCO₃ solution (30 mL). The organic layer wasdried over Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified through flash chromatography on silica gel (1:19MeOH:CH₂Cl₂) to afford the entitled product (0.8348 g, 70%) as a yellowgel. ¹H NMR (400 MHz, CDCl₃) δ 8.90 (s, 1H), 8.72 (s, 1H), 7.85 (d,J=8.5 Hz, 1H), 7.54 (dd, J=8.7, 5.3 Hz, 2H), 7.17 (t, J=8.6 Hz, 2H),6.81 (d, J=8.5 Hz, 1H), 4.15 (t, J=9.8 Hz, 1H), 3.53 (d, J=13.5 Hz, 1H),3.47-3.20 (m, 3H), 2.05 (dt, J=17.2, 8.5 Hz, 1H), 1.96-1.80 (m, 2H),1.71 (s, 1H), 1.44 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 164.3, 164.0 (d,J=252.0 Hz, 157.5, 144.4, 142.6, 138.1 (d, J=8.7 Hz), 131.8, 131.4,127.9, 125.7 (d, J=3.7 Hz), 124.3, 117.5 (d, J=22.0 Hz), 80.7, 56.0,47.7, 47.4, 29.6, 28.4, 24.0. MS (ESI) m/z 376.1 (100%, [M+H-Boc]⁺).

4-(4-fluorophenyl)thio)-N-((1-methylpyrrolidin-2-yl)methyl)-3-nitrobenzamide(WW3-107)

A mixture of WW3-79 (0.3116 g, 0.66 mmol) and HCl (4 M in 1,4-dioxane,12 mL) was stirred at room temperature for 2.5 h and then was slowlypoured into 4 N NaOH (35 mL) solution. The resulting solution wasextracted with CH₂Cl₂ (3×35 mL). The combined organic layers were driedover Na₂SO₄, filtered and concentrated under reduced pressure to affordthe deprotected amine which was used directly for next step withoutfurther purification.

A mixture of the deprotected amine above, formaldehyde (37%, 0.1914 g,2.36 mmol), AcOH (0.05 mL, 0.87 mmol), CH₃OH (12 mL) and NaBH(OAc)₃(0.3188 g, 1.47 mmol) was stirred at room temperate for 17 h. Thereaction solution was then slowly poured into a solution of saturatedaq. NaHCO₃ (35 mL) and extracted with DCM (3×35 mL). The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified through flash chromatographyon silica gel (1:19 MeOH:CH₂Cl₂) to afford the title product (0.1837 g,72% two steps) as a yellow solid. mp 101-103° C.; ¹H NMR (400 MHz,CDCl₃) δ 8.65 (s, 1H), 7.80 (dd, J=8.4, 2.1 Hz, 1H), 7.60-7.46 (m, 2H),7.26-7.13 (m, 3H), 6.84 (d, J=8.5 Hz, 1H), 3.76 (ddd, J=14.0, 7.6, 3.1Hz, 1H), 3.32 (d, J=13.7 Hz, 1H), 3.14 (s, 1H), 2.60 (s, 1H), 2.38 (s,3H), 2.37-2.25 (m, 1H), 1.94 (dq, J=12.6, 8.1 Hz, 1H), 1.81-1.57 (m,3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.9, 164.06 (d, J=252.4 Hz), 144.3,143.1, 138.2 (d, J=8.7 Hz), 131.5, 128.1, 125.4 (d, J=3.7 Hz), 124.6,117.7 (d, J=22.0 Hz), 64.4, 57.1, 40.5, 40.0, 28.0, 22.9. MS (ESI) m/z390.1 (100%, [M+H]⁺).

N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)-3-nitro-N-(prop-2-yn-1-yl)benzamide(WW2-292)

DS-1-033 (0.1355 g, 0.34 mmol) was dissolved in anhydrous DMF (2.0 mL),followed by the addition of NaH (60%, 0.0272 g, 0.68 mmol) at 0° C. Theflask was then immediately flushed with argon and sealed with a rubberseptum fitted with an argon balloon. The reaction solution was stirredfor 8 min at 0° C. and propargyl bromide (80% in toluene, 0.041 mL, 0.37mmol) was added via syringe. The resulting solution was stirred for 3 hat 0° C. and the reaction was quenched by slowly adding water (25 mL)and then EtOAc (25 mL). The resulting bi-phase solution was washed by20% LiCl solution (3×25 mL) and brine (25 mL). The organic layer wasdried over Na₂SO₄, filtered and concentrated under reduced pressure. Theflash chromatography on silica gel (1:19 Methanol:CH₂Cl₂) provided thedesired product as a mixture of two rotamers as a brown oil (0.0722 g,49%). The rotamers coalesced at 50° C. ¹H NMR (400 MHz, CDCl₃, 50° C.) δ8.45 (s, 1H), 7.62-7.52 (m, 2H), 7.46 (dd, J=8.4, 1.9 Hz, 1H), 7.22-7.11(m, 2H), 6.84 (d, J=8.4 Hz, 1H), 4.30 (s, 2H), 3.49 (d, J=51.7 Hz, 2H),3.05 (t, J=9.7 Hz, 1H), 2.91-2.64 (m, 2H), 2.43-2.23 (m, 2H), 2.18 (td,J=9.2, 7.2 Hz, 1H), 1.86 (dq, J=12.3, 8.2 Hz, 1H), 1.75-1.39 (m, 3H),1.04 (t, J=7.2 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃, 50° C.) δ 169.1, 164.0(d, J=252.3 Hz), 144.5, 141.4, 138.0 (d, J=8.6 Hz), 132.7, 132.0, 128.1,125.7 (d, J=3.7 Hz), 124.8, 117.5 (d, J=22.1 Hz), 78.4, 73.5, 62.8,53.3, 49.3, 28.9, 22.7, 12.8. MS (ESI) m/z 442.2 (100%, [M+H]⁺).

tert-butyl (2-(ethyl(2-hydroxyethyl)amino)ethyl)carbamate

2-(Ethylamino)ethanol (0.9173 g, 10.31 mmol) and tert-butyl(2-bromoethyl)carbamate (2.4964 g, 11.14 mmol) were dissolved in CH₃CN(12 mL), followed by addition of potassium carbonate (2.0617 g, 14.84mmol). The formed suspension was stirred vigorously at 50° C. for 20 hand then filtered through a pad of Celite, washed by methanol. Uponremoval of the solvents in vacuo, the residue was dissolved in DCM (3mL) and purified through a short pad of silica gel, eluted with amixture of 19:1 DCM/methanol to afford the entitled product (2.1548 g,90%) as a colorless gel. ¹H NMR (400 MHz, CDCl₃) δ 3.55 (t, J=5.3 Hz,2H), 3.17 (q, J=5.7 Hz, 2H), 2.69-2.49 (m, 6H), 1.41 (s, 9H), 1.01 (t,J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 156.2, 58.8, 55.3, 53.0, 47.7,40.6, 38.5, 28.3, 11.5. MS (ESI) m/z 233.2 (100%, [M+H]⁺).

2-(ethyl(2-(4-((4-fluorophenyl)thio)-3-nitrobenzamido)ethyl)amino)ethyl4-((4-fluorophenyl)thio)-3-nitrobenzoate

A mixture of tert-butyl (2-(ethyl(2-hydroxyethyl)amino)ethyl)carbamate(0.2921 g, 1.26 mmol), HCl (37%, 2 mL) and 1,4-dioxane (6 mL) wasstirred at room temperature for 4 hours. Upon removal of the excess HCland solvents, the residue was neutralized by 2N NaOH solution, extractedwith DCM (3×30 mL). The combined organic layers were dried over Na₂SO₄,filtered and concentrated under reduced pressure to about 6 mL.

To a flame-dried flask equipped with a reflux condenser were added4-((4-fluorophenyl)thio)-3-nitrobenzoic acid (0.2970 g, 1.01 mmol) andthionyl chloride (6 mL). The resulting reaction solution was stirred atreflux for 4 h. After being cooled down to room temperature, the excessthionyl chloride was evaporated under reduced pressure to afford theacyl chloride intermediate as a yellow solid.

To the deprotected amine above in DCM solution was added Et₃N (0.30 mL,2.16 mmol) at 0° C., followed by addition of the acyl chlorideintermediate in portions in 5 min. The resulting solution was stirred at0° C. for 1 h and then at room temperature for one more hour. Thereaction solution was transferred to a separatory funnel and 25 mLsaturated NaHCO₃ solution was added. The resulting bi-phase solution wasextracted with DCM (3×25 mL) and the combined organic layers were driedover Na₂SO₄, filtered and concentrated under reduced pressure. The flashchromatography on silica gel (1:19 Methanol:CH₂Cl₂) provided theentitled product as a yellow gel (0.2529 g, 37%). ¹H NMR (400 MHz,CDCl₃) δ 8.62 (s, 1H), 8.48 (s, 1H), 7.76 (d, J=8.6 Hz, 1H), 7.66 (d,J=8.5 Hz, 1H), 7.61-7.49 (m, 4H), 7.20-7.10 (m, 5H), 6.81-6.68 (m, 2H),4.39 (t, J=5.3 Hz, 2H), 3.45 (q, J=5.4 Hz, 2H), 2.83 (t, J=5.3 Hz, 2H),2.71 (t, J=5.8 Hz, 2H), 2.59 (q, J=7.1 Hz, 2H), 0.99 (t, J=7.0 Hz, 3H).¹³C NMR (100 MHz, CDCl₃) δ 164.4, 164.3, 164.1 (d, J=252.4 Hz). 164.0(d, J=252.2 Hz), 145.5, 145.4, 143.8, 143.1, 138.2 (d, J=8.6 Hz), 138.2(d, J=8.7 Hz), 133.2, 131.6, 131.3, 127.8, 126.9, 126.8, 125.3 (d, J=3.4Hz), 124.9 (d, J=3.5 Hz), 124.2, 117.8 (d, J=22.1 Hz). 117.6 (d, J=22.0Hz), 63.7, 52.5, 52.3, 48.2, 37.6, 12.2. MS (ESI) m/z 683.2 (30%,[M+H]+).

N-(2-(ethyl(2-hydroxyethyl)amino)ethyl)-4-((4-fluorophenyl)thio)-3-nitrobenzamide(WW3-62)

To a 15 mL flask were added2-(ethyl(2-(4-((4-fluorophenyl)thio)-3-nitrobenzamido)ethyl)amino)ethyl4-((4-fluorophenyl)thio)-3-nitrobenzoate (0.1262 g, 0.18 mmol), water (3mL), NaOH (0.0905 g, 2.26 mmol), THF (1 mL) and methanol (1 mL). Thereaction solution was stirred at 50° C. for 3 h and was diluted withwater (20 mL), followed by extraction with DCM (3×25 mL). The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to afford the desired product as a yellow gel (0.0507g, 67%). ¹H NMR (400 MHz, CDCl₃) δ 8.64 (d, J=1.9 Hz, 1H), 7.79 (dd,J=8.6, 2.0 Hz, 1H), 7.63-7.44 (m, 3H), 7.17 (t, J=8.6 Hz, 2H), 6.78 (d,J=8.5 Hz, 1H), 3.61 (t, J=5.2 Hz, 2H), 3.47 (q, J=5.6 Hz, 2H), 2.91 (s,1H), 2.69 (t, J=5.8 Hz, 2H), 2.67-2.55 (m, 4H), 0.99 (t, J=7.1 Hz, 3H).¹³C NMR (100 MHz, CDCl₃) δ 164.7, 164.0 (d, J=250.9 Hz), 144.0, 143.0,143.0, 138.1 (d, J=8.6 Hz), 131.9, 131.5, 127.91, 125.4 (d, J=3.7 Hz),124.3, 117.6 (d, J=22.0 Hz), 59.5, 55.1, 52.0, 48.0, 38.4, 11.6. MS(ESI) m/z 408.2 (100%, [M+H]⁺).

3-azido-N-((1-ethylpyrrolidin-2-yl)methyl)-4-((4-fluorophenyl)thio)benzamide(WW3-57)

To a 25 mL flask containing DS-1-225 (0.1423 g, 0.38 mmol) were added 6M HCl (1.5 ml), THF (1.5 ml) and DMF (0.8 ml). The mixture was cooled to0° C. and NaNO₂ (0.0346 g, 0.50 mmol) was added. The reaction solutionwas stirred for 50 min at 0° C. and NaN₃ (0.0398 g, 0.61 mmol) in water(0.6 mL) was added. The resulting solution was then allowed to warm toroom temperature and stirred overnight, followed by addition of 1 N NaOH(30 mL). The solution was extracted with CH₂Cl₂ (3×30 ml) and thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated. The residue was dissolved in EtOAc (30 mL), washed by 10%LiCl solution (30 mL) and brine (3×30 mL) to afford the entitled product(0.1298 g, 85%) as light brown gel. ¹H NMR (400 MHz, CDCl₃) δ 7.91 (s,1H), 7.75 (d, J=1.8 Hz, 1H), 7.52-7.39 (m, 3H), 7.14-7.04 (m, 2H), 6.76(d, J=8.3 Hz, 1H), 3.73 (ddd, J=14.3, 7.1, 5.3 Hz, 1H), 3.60-3.36 (m,2H), 3.13 (s, 1H), 3.03-2.90 (m, 1H), 2.71-2.35 (m, 2H), 2.11-1.94 (m,1H), 1.91-1.81 (m, 2H), 1.75 (dt, J=12.6, 6.4 Hz, 1H), 1.23 (t, J=7.2Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 166.2, 163.3 (d, J=250.2 Hz), 137.3,136.7 (d, J=8.4 Hz), 133.9, 132.5, 128.0, 126.1 (d, J=3.5 Hz), 123.3,117.0 (d, J=22.0 Hz), 116.9, 64.6, 53.7, 49.7, 40.4, 27.9, 23.3, 12.4.MS (ESI) m/z 400.2 (100%, [M+H]⁺).

Ethyl 4-((4-fluorophenyl)thio)-3-nitrobenzoate

A mixture of 4-((4-fluorophenyl)thio)-3-nitrobenzoic acid (0.8795 g,3.00 mmol), DMAP (0.0814 g, 0.67 mmol), EDC hydrochloride (0.6330 g,3.30 mmol) and CH₂Cl₂ (7 mL) was stirred at room temperature for 30 min.Ethanol (0.7102 g, 15.44 mmol) was then added and the resulting solutionwas stirred at room temperature for 24 h. The reaction solution wasdiluted with CH₂Cl₂ (30 mL), washed by brine (30 mL) and 2 N NaOHsolution (30 mL). The organic layer was dried over Na₂SO₄, filtered andconcentrated under reduced pressure. The residue was purified throughflash chromatography on silica gel (10:1 Hexane:EtOAc) to afford thetitle product as yellow solid (0.8088 g, 84%). mp 112-115° C.; ¹H NMR(400 MHz, CDCl₃) δ 8.83 (d, J=1.6 Hz, 1H), 7.94 (dd, J=8.6, 1.9 Hz, 1H),7.69-7.44 (m, 2H), 7.26-7.12 (m, 2H), 6.84 (d, J=8.6 Hz, 1H), 4.37 (q,J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ164.3,164.10 (d, J=252.4 Hz), 145.0, 144.3, 138.2 (d, J=8.7 Hz), 133.5, 127.8,127.6, 127.0, 125.3 (d, J=3.6 Hz), 117.7 (d, J=22.0 Hz), 61.7, 14.2.

Ethyl 3-amino-4-((4-fluorophenyl)thio)benzoate

To a 50 mL flask were added ethyl4-((4-fluorophenyl)thio)-3-nitrobenzoate (0.2132 g, 0.66 mmol), methanol(5 mL) and Pd/C (2 spatula, 10% on active carbon). The reaction flaskwas sealed by a septum and after the removal of air using vacuum, ahydrogen balloon was fitted on the top of the septum. The reactionsuspension was then stirred at room temperature for 15 h and wasfiltered through a pad of Celite, washed by methanol. The filtrate wasconcentrated under reduced pressure to provide the desired product(0.1856 g, >95%) as a yellow gel. ¹H NMR (400 MHz, CDCl₃) δ 7.45 (s,1H), 7.38 (s, 2H), 7.15 (dd, J=8.8, 5.1 Hz, 2H), 6.96 (t, J=8.7 Hz, 2H),4.36 (q, J=7.1 Hz, 2H), 1.38 (t, J=7.1 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 166.3, 161.7 (d, J=246.3 Hz), 147.6, 135.6, 132.2, 130.1 (d, J=8.0Hz), 129.4, 121.2, 119.3 (d, J=2.2 Hz), 116.3 (d, J=22.2 Hz), 116.1,61.1, 14.3. MS (ESI) m/z 292.1 (100%, [M+H]⁺).

3-azido-4-((4-fluorophenyl)thio)benzoic acid

A mixture of ethyl 3-amino-4-((4-fluorophenyl)thio)benzoate (0.1674 g,0.58 mmol), LiOH.H₂O (0.1496 g, 56%, 1.99 mmol), THF (5 mL) and water (3mL) was stirred at 72° C. for 2.5 h. The reaction solution was cooleddown to room temperature and then 0° C. in an ice-water bath. Thereaction solution was then acidified to pH=1 using 6 N HCl solution andNaNO₂ (0.0505 g, 0.73 mmol) was added at 0° C. The resulting solutionwas stirred for 25 min at 0° C. and NaN₃ (0.0623 g, 0.96 mmol) wasadded. The reaction solution was then allowed to warm up to roomtemperature gradually and stirred overnight. The reaction mixture wasdiluted with 15 mL water and extracted with CH₂Cl₂ (3×25 ml). Thecombined organic layers were dried by Na₂SO₄, filtered and concentratedto afford the entitled product (0.1322 g, 79% two steps) as orangesolid. mp 156-158° C.; ¹H NMR (400 MHz, CDCl₃) δ 7.82 (d, J=1.7 Hz, 1H),7.65 (dd, J=8.3, 1.7 Hz, 1H), 7.52 (dd, J=8.6, 5.3 Hz, 2H), 7.15 (t,J=8.6 Hz, 2H), 6.73 (d, J=8.3 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 170.9,163.6 (d, J=251.5 Hz), 137.9, 137.4 (d, J=8.6 Hz), 136.5, 127.2, 126.9,126.8, 125.1 (d, J=3.5 Hz), 119.3, 117.3 (d, J=22.0 Hz). MS (ESI) m/z288.0 (100%, [M−H]⁻).

3-azido-N-(2-(ethyl(prop-2-yn-1-yl)amino)ethyl)-4-((4-fluorophenyl)thio)benzamide(WW3-77)

A mixture of 3-azido-4-((4-fluorophenyl)thio)benzoic acid (0.1231 g,0.43 mmol), DMAP (0.0153 g, 0.13 mmol), EDC hydrochloride (0.0886 g,0.46 mmol) and CH₂Cl₂ (3 mL) was stirred at room temperature for 20 min.N¹-ethyl-N¹-(prop-2-yn-1-yl)ethane-1,2-diamine (0.0520 g, 0.41 mmol) inDCM (1 mL) was then added and the resulting solution was stirred at roomtemperature for 20 h. The reaction solution was diluted with CH₂Cl₂ (25mL), washed by brine (25 mL) and saturated NaHCO₃ solution (25 mL). Theorganic layer was dried over Na₂SO₄, filtered and concentrated underreduced pressure. The residue was purified through flash chromatographyon silica gel (1:19 MeOH:CH₂Cl₂) to afford the title product as orangegel (0.1364 g, 83%). ¹H NMR (400 MHz, CDCl₃) δ 7.61 (d, J=1.7 Hz, 1H),7.45 (dd, J=8.8, 5.2 Hz, 2H), 7.25 (dd, J=8.2, 1.8 Hz, 1H), 7.10 (t,J=8.6 Hz, 2H), 6.85 (s, 1H), 6.75 (d, J=8.2 Hz, 1H), 3.48 (dd, J=11.4,4.9 Hz, 2H), 3.40 (d, J=2.4 Hz, 2H), 2.74 (dd, J=6.4, 5.3 Hz, 2H), 2.58(q, J=7.2 Hz, 2H), 2.19 (t, J=2.3 Hz, 1H), 1.05 (t, J=7.2 Hz, 3H). ¹³CNMR (100 MHz, CDCl₃) δ 165.9, 163.3 (d, J=250.2 Hz), 137.3, 136.6 (d,J=8.4 Hz), 133.8, 133.3, 128.1, 126.2 (d, J=3.4 Hz), 123.0, 117.5, 117.0(d, J=22.1 Hz), 78.2, 73.3, 51.4, 47.3, 41.2, 37.1, 12.7. MS (ESI) m/z398.2 (100%, [M+H]⁺).

2-((2-((tert-butoxycarbonyl)amino)ethyl)(prop-2-yn-1-yl)amino)ethyl4-benzoylbenzoate

A mixture of 4-benzoylbenzoic acid (0.2009 g, 0.89 mmol), DMAP (0.0344g, 0.28 mmol), EDC hydrochloride (0.1758 g, 0.92 mmol) and CH₂Cl₂ (5 mL)was stirred at room temperature for 1 h. tert-Butyl(2-((2-hydroxyethyl)(prop-2-yn-1-yl)amino)ethyl)carbamate (0.1952 g,0.81 mmol) in CH₂Cl₂ (2 mL) was then added and the resulting solutionwas stirred at room temperature for 18.5 h. The reaction solution wasdiluted with CH₂Cl₂ (25 mL), washed by brine (30 mL) and saturatedNaHCO₃ solution (30 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified through flash chromatography on silica gel (1:19 MeOH:CH₂Cl₂)to afford the title product (0.3031 g, 83%) as colorless gel. ¹H NMR(400 MHz, CDCl₃) δ 8.14 (d, J=8.3 Hz, 2H), 7.82 (d, J=8.2 Hz, 2H), 7.78(d, J=7.1 Hz, 2H), 7.60 (t, J=7.4 Hz, 1H), 7.48 (t, J=7.7 Hz, 2H), 4.43(t, J=5.5 Hz, 2H), 3.48 (d, J=2.4 Hz, 2H), 3.21 (q, J=5.7 Hz, 2H), 2.94(t, J=5.6 Hz, 2H), 2.71 (t, J=5.9 Hz, 2H), 2.21 (t, J=2.3 Hz, 1H), 1.38(s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 195.9, 165.8, 155.9, 141.3, 136.9,133.1, 132.9, 130.1, 129.8, 129.5, 128.4, 78.1, 73.5, 63.2, 52.8, 51.9,42.4, 37.8, 28.8, 28.4. MS (ESI) m/z 451.2 (100%, [M+H]⁺).

2-((2-(4-((4-fluorophenyl)thio)-3-nitrobenzamido)ethyl)(prop-2-yn-1-yl)amino)ethyl4-benzoylbenzoate (WW3-80)

2-((2-((tert-Butoxycarbonyl)amino)ethyl)(prop-2-yn-1-yl)amino)ethyl4-benzoylbenzoate (0.1818 g, 0.40 mmol) was dissolved in anhydrousCH₂Cl₂ (2.5 mL), followed by addition of trifluoroacetic acid (1 mL,13.06 mmol). The resulting solution was stirred at room temperature for3 h and then was slowly poured into 30 mL saturated NaHCO₃ solution at0° C. The bi-phasic solution was extracted with CH₂Cl₂ (3×30 mL). Thecombined organic layers were dried over Na₂SO₄, filtered andconcentrated under reduced pressure to afford the deprotected aminewhich was used directly for next step without further purification.

A mixture of 4-((4-fluorophenyl)thio)-3-nitrobenzoic acid (0.1191 g,0.41 mmol), DMAP (0.0112 g, 0.09 mmol), EDC hydrochloride (0.0900 g,0.47 mmol) and CH₂Cl₂ (2 mL) was stirred at room temperature for 20 min.The deprotected amine above in CH₂Cl₂ (3 mL) was then added and theresulting reaction mixture was stirred at room temperature overnight andwas diluted with CH₂Cl₂ (25 mL), washed by brine (25 mL) and saturatedNaHCO₃ solution (25 mL). The organic layer was dried over Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified through flash chromatography on silica gel (1:19 MeOH:CH₂Cl₂)to afford the title product (0.1844 g, 73% two steps) as yellow gel. ¹HNMR (400 MHz, CDCl₃) δ 8.58 (s, 1H), 7.97 (d, J=8.3 Hz, 2H), 7.84-7.64(m, 5H), 7.64-7.58 (m, 1H), 7.56-7.44 (m, 4H), 7.23-7.05 (m, 3H), 6.75(d, J=8.6 Hz, 1H), 4.49 (t, J=5.2 Hz, 2H), 3.68-3.46 (m, 4H), 3.01 (t,J=5.1 Hz, 2H), 2.91 (t, J=5.7 Hz, 2H), 2.27 (t, J=2.3 Hz, 1H). ¹³C NMR(100 MHz, CDCl₃) δ 195.7, 165.9, 164.6, 164.0 (d, J=252.4 Hz), 144.0,143.1, 141.3, 138.1 (d, J=8.7 Hz), 136.7, 133.0, 132.8, 131.7, 131.3,130.0, 129.7, 129.3, 128.5, 127.8, 125.3 (d, J=3.5 Hz), 124.4, 117.6 (d,J=22.1 Hz), 77.7, 74.1, 62.9, 52.5, 52.2, 43.0, 37.3. MS (ESI) m/z 626.2(100%, [M+H]⁺).

TABLE A Exemplary analog compounds and their properties. EC50 MW (TD-TPSA (g/ CNS NSC) S9 Structure CLogP CLogD (Å) mol) HBD pKa MPO (nM)T_(1/2)

3.91 3.91 85.1 383 1 4.14 4.3 >1000

4.16 3.08 75.5 403 1 8.45 4.2 33 113.6

4.16 3.08 75.5 403 1 8.45 4.2 45 119

4.05 2.4 75.5 391 1 9.04 4.4 22 45

4.16 3.08 75.5 403 1 8.45 4.2 31 66

4.02 2.93 75.5 385 1 8.45 4.5 50 35.4

3.86 1.98 75.5 403 1 9.29 4.4 79 105.0

3.74 2.9 75.5 389 1 8.17 4.7 52 187.3

4.90 3.81 75.5 453 1 8.45 3.1 86

4.3 3.22 75.5 421 1 8.45 3.9 118

4.16 3.08 75.5 403 1 8.45 4.2 88 35.2

3.39 2.31 88.4 386 1 8.44 5.1 419

4.16 3.08 75.5 403 1 8.45 4.2 102

3.26 2.17 204.6 443 2 8.45 4.0 >1000

4.64 2.67 58.4 389 1 9.30 3.8 36 21.4

3.42 0.18 84.3 375 2 10.56 4.2 132 .240

3.12 3.11 84.7 405 1 5.85 4.9 25

3.47 2.39 84.7 387 1 8.45 5.0 36

4.71 3.58 87.5 386 2 8.50 3.4 255

4.22 2.97 32.3 358 1 8.62 4.0 394

2.02 / 72.2 419 1 −1.23 4.4 >1000

5.26 4.18 75.5 428 1 8.45 3.1 516

4.30 3.22 75.5 421 1 8.45 3.9 51 87.7

2.80 1.71 88.4 386 1 8.45 5.4 150 92.4

4.22 3.13 75.5 418 1 8.45 4.0 35 96.3

3.86 2.78 84.7 416 1 8.45 4.4 62 48.1

2.79 2.29 92.6 419 1 7.73 5.2 >1000

3.96 2.87 118.6 430 1 8.45 3.2 69

4.05 2.96 75.5 392 1 8.45 4.4 192

4.62 3.54 75.5 420 1 8.45 3.6 51 88.8

3.19 2.10 101.5 400 2 8.45 4.5 340

0.95 0.91 112.0 429 2 8.45 4.0 >1000

3.62 2.52 75.5 371 1 8.47 4.9 >1000

3.62 2.52 75.5 371 1 8.46 5.0 >1000

4.79 3.70 75.5 464 1 8.45 3.1 62

4.16 3.08 75.5 403 1 8.45 4.2 95

2.54 0.53 101.5 392 2 9.51 4.2 >1000

2.90 1.13 147.7 619 4 7.70 3.0 ≈40000

3.60 2.46 45.2 359 1 8.50 5.1 >1000

1.12 1.18 121.7 450 2 8.46 3.6 >1000

3.39 3.39 4.08 3.07 4.05 3.12 4.09 4.52 2.63 2.09 2.89 1.89 2.40 3.113.00 3.44 95.7 58.4 56.1 75.4 75.5 84.7 84.7 101.8 401 373 383 402 391405 440 476 2 2 1 2 1 1 1 1 8.37 8.68 8.56 8.55 9.04 5.85 8.45 8.45 4.34.8 4.4 4.9 4.4 4.9 4.0 2.9 485 192 53 >1000 59 44 269 >1000     65

4.17 3.09 88.4 437 1 8.45 3.9 535

4.53 3.45 75.5 400 1 8.45 3.8 55

3.74 3.74 81.5 376 1 −1.07 4.5 >1000

4.27 3.18 5.09 5.23 4.62 5.04 2.92 3.05 4.06 4.14 3.54 3.96 66.7 84.772.7 75.5 75.5 75.5 415 419 404 454 420 414 0 1 0 1 1 1 8.73 6.95 8.398.45 8.45 8.45 4.1 4.8 3.5 2.9 3.6 3.2 58 42 >1000 93 70 169 26.7 93.6

3.06 2.89 66.5 437 1 7.09 4.8 236 >240

4.53 3.45 75.5 400 1 8.45 3.8 86

3.39 2.20 65.4 367 1 8.56 5.2 117.5

3.04 3.02 65.4 386 1 5.96 5.1 40.1

3.92 2.35 2.99 4.36 3.33 2.33 1.91 3.18 78.2 74.6 104.4 32.3 401 369 401376 1 1 2 1 7.90 5.96 8.45 8.56 4.4 5.6 4.5 3.8 49 74 564 87   99   41

3.92 3.50 101.2 440 1 7.60 3.7 155

3.43 2.94 101.2 399 1 7.72 4.5 233

4.38 3.15 75.4 418 0 8.61 4.0 15

4.09 3.47 132.9 424 1 7.90 3.1 >1000

6.81 6.47 110.5 594 1 7.47 2.2 >1000

4.55 3.93 110.5 472 1 7.90 2.6 >1000

4.55 3.93 110.5 472 1 7.90 2.6 >1000

3.19 2.98 101.2 397 1 7.20 4.6 >1000

3.24 4.16 1.02 3.04 102.2 84.2 399 403 2 1 9.66 8.49 3.9 4.2 >1000 60

3.18 2.32 87.4 418 1 8.20 5.1 287

4.19 3.44 84.2 404 1 8.06 4.2 50

4.08 4.07 113.7 505 1 5.78 2.5 Not active

2.80 0.99 96.2 404 2 9.22 4.4 141

5.50 4.41 84.2 488 1 8.45 2.7 129

4.90 3.79 84.2 454 1 8.47 3.1

3.19 2.09 110.2 401 2 8.47 4.2 301

4.62 3.54 84.2 420 1 8.45 3.6 119

4.53 3.45 84.2 400 1 8.45 3.8 78

3.86 2.78 93.4 416 1 8.45 4.3 119

5.23 4.14 84.2 454 1 8.45 2.9 68

5.23 4.14 84.2 454 1 8.45 2.9 510

4.77 3.69 96.5 437 1 8.44 3.1 125

3.71 2.66 104.4 401 2 8.73 3.7 139

3.71 2.66 104.4 401 2 8.23 3.9 160

3.86 2.78 93.4 415.5 1 8.45 4.3 185

3.25 2.17 104.4 415.5 2 8.45 4.2 154

5.47 3.45 75.4 441.5 0 8.54 3.4 531

6.07 3.57 118.1 491.5 1 8.48 1.9 >1000

5.65 3.35 81.1 399.5 1 8.55 3.6 63

4.11 1.90 104.4 407.5 2 8.48 3.9 57

5.62 3.67 81.1 397.5 1 7.90 3.7 239

7.95 6.81 127.5 625.7 1 6.10 1.8 161

Plasma Stability

Compounds were diluted to a final concentration of 2 μM in commercialplasma (Bioreclamation, Westbury, N.Y.) and 200 μl aliquoted intomultiple Eppendorf vials. One vial was immediately extracted with theaddition of an equal volume of methanol containing 0.2% formic acid and200 ng/ml of an internal standard (either n-benzylbenzamide ortolbutamide, Sigma, St. Louis, Mo.). The vial was vortexed for 15seconds, incubated 10 min at RT and centrifuged at 16,100×g for 5 min.The supernatant was re-centrifuged 5 min 16,100×g and the secondarysupernatant placed in an HPLC vial and analyze by LC-MS/MS as describedabove. The remaining aliquots were incubated in a 37° C. water bath forup to 24 hours. At various points, samples were removed and processed asdescribed above. LC-MS/MS analyses was as described for the S9 andhepatocyte stability analyses.

Metabolic Stability Data Analysis

The method described in McNaney, et al (McNaney, C A, D M Drexler, SHnatyshyn, T A Zvyaga, J O Knipe, J V Belcastro, and M Sanders. 2008. Anautomated liquid chromatography-mass spectrometry process to determinemetabolic stability half-life and intrinsic clearance of drug candidatesby substrate depletion. Assay and Drug Development Technologies6:121-129. Determination of Plasma and Brain Pharmacokinetics) was usedwith modification for determination of metabolic stability half-life bysubstrate depletion. A “% remaining” value was used to assess metabolicstability of a compound over time. The LC/MS/MS peak area of theincubated sample at each time point was divided by the LC/MS/MS peakarea of the time 0 (T0) sample and multiplied by 100. The natural Log(LN) of the % remaining of compound was then plotted versus time (inmin) and a linear regression curve plotted going through y-intercept atLN(100). The metabolism of some compounds failed to show linear kineticsat later time point, so those time points were excluded. The half-life(T½) was calculated as T½=0.693/slope.

Determination of Plasma and Brain Pharmacokinetics

Compounds were prepared for dosing by dissolving in DMSO at 25-50 mg/ml.The compounds were diluted to the final formulation as listed in theindividual Excel data files (most often 10% DMSO/10% cremophor EL/80%D5W (5% dextrose in water, pH 7.4).) Adult CD-1 female mice were dosedIP in a total volume of 0.2 ml and at varying times post-dose weresacrificed by inhalation overdose of CO2. Whole blood was collected bycardiac puncture with an ACD solution (sodium citrate) coated syringeand needle. The blood was subsequently centrifuged at 9300×g for 10′ toisolate plasma. Plasma was stored at −80° C. until analysis. Brains wereisolated from mice immediately after sacrifice, rinsed three times withPBS and blotted gently to remove any surface adhering blood, weighed,and snap frozen in liquid nitrogen. Lysates were prepared byhomogenizing the brain tissue in a 3-fold volume of PBS (weight of brainin g X=volume of PBS in ml added). Total lysate volume was estimated asvolume of PBS added+volume of brain in ml. One hundred μl of eitherplasma or brain was processed by addition of 200 μl of acetonitrile ormethanol containing 0.15% formic acid and 150 ng/ml internal standard(n-benzyl benzamide or tolbutamide) to precipitate plasma or tissueprotein and release bound drug. This mixture was centrifuged at 16,100×gfor 5 min, the supernatant was re-centrifuged and analyzed directly byLC-MS/MS

Compound levels were monitored by LC-MS/MS using an AB/Sciex (AppliedBiosystems, Foster City, Calif.) 4000 Qtrap mass spectrometer coupled toa Shimadzu Prominence LC. The compound was detected with the massspectrometer in MRM (multiple reaction monitoring) mode by following theprecursor to fragment ion transitions indicated in the Methods box forindividual data files. Standard curves were prepared by addition ofcompound to blank plasma or blank brain lysate. A value of 3× above thesignal obtained from blank plasma or brain lysate was designated thelimit of detection (LOD). The limit of quantitation (LOQ) was defined asthe lowest concentration at which back calculation yielded aconcentration within 20% of theoretical. In general back calculation ofpoints on both curves yielded values within 15% of theoretical over 4orders of magnitude (1000 to 1 ng/ml). Pharmacokinetic parameters werecalculated using the noncompartmental analysis tool of Phoenix WinNonLin(Certara, Corporation, Princeton, N.J.). The amount of compound in thevasculature of the brain was subtracted using reference values for theamount of blood in the brain (30 μl/g brain tissue) and the measuredplasma concentration (Kwon, Y. (2001). The Handbook of EssentialPharmacokinetics, Pharmacodynamics, and Drug Metabolism for IndustrialScientists. Kluwer Academic/Plenum Publishers, pp 231-232).

Compound was assumed to partition equally between plasma and blood forthis determination. The Brain:Blood ratio was calculated using thesubtracted brain AUC and the plasma AUC.

TABLE B pharmacokinetic parameters for exemplary compounds.Bioavailability (IP) in Plasma Bioavailability (IP) in Brain AUC CI AUCCI min * ng/mL mil/min min * ng/mL ml/min Cmax T_(1/2) (10 mg/kg (10mg/kg Cmax T_(1/2) (10 mg/kg (10 mg/kg Compound (ng/ml) (min) IP) IP)(ng/ml) (min) IP) IP) A 7946.67 101.97 513,117.5 — 52.08 175.36 17,465.9B 1387 52 139,721 1.66 646 62 95,916 2.38 C 7946.67 101.97 513,117.490.47 52.08 175.36 17,465.90 13.85 D 2160 137 309,840 0.75 503 240130,384 1.75 E 2833.33 83.6 163.543 1.47 1035.00 118.4 49,878 4.68 F1103 52 50,857 4.51 1701 56 206,079 1.11

Example 2. Deregulators of Cholesterol Biosynthesis Pathways

Microarray analysis was conducted to identify overrepresented pathwaysassociated with treatment with compound 4C12 for 48 hr. The results,which are shown in FIG. 9, suggest that compound 4C12 deregulates thecholesterol biosynthesis pathway.

Cholesterol Pathways

The major types of lipids that circulate in plasma include cholesteroland cholesteryl esters, phospholipids and triglycerides. Braunwald'sHeart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8^(th)Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1071.Cholesterol contributes an essential component of mammalian cellmembranes and furnishes substrate for steroid hormones and bile acids.Many cell functions depend critically on membrane cholesterol, and cellstightly regulate cholesterol content. Most of the cholesterol in plasmacirculates in the form of cholesteryl esters in the core of lipoproteinparticles. The enzyme lecithin cholesterol acyl transferase (LCAT) formscholesteryl esters in the blood compartment by transferring a fatty acylchain from phosphatidylcholine to cholesterol. Id.

Lipoproteins are complex macromolecular structures composed of anenvelope of phospholipids and free cholesterol, a core of cholesterylesters and triglycerides. Id. at 1072. Triglycerides consist of athree-carbon glycerol backbone covalently linked to three fatty acids.Their fatty acid composition varies in terms of chain length and degreeof saturation. Triglyceride molecules are nonpolar and hydrophobic, andare transported in the core of the lipoprotein. Hydrolysis oftriglycerides by lipases generates free fatty acids (FFAs) used forenergy. Id. Phospholipids, constituents of all cellular membranes,consist of a glycerol molecule linked to two fatty acids. The fattyacids differ in length and in the presence of a single or multipledouble bonds. The third carbon of the glycerol moiety carries aphosphate group to which one of four molecules is linked: choline(phosphatidylcholine or lecithin), ethanolamine(phosphatidylethanolamine), serine (phosphatidylserine), or inositol(phosphatidylinositol). Phospholipids, which are polar molecules, moresoluble than triglycerides or cholesterol or its esters, participate insignal transduction pathways. Hydrolysis by membrane-associatedphospholipases generates second messengers such as diacyl glycerols,lysophospholipids, phoshatidic acids and free fatty acids (FFAs) such asarachidonate that can regulate many cell functions. Id.

The apolipoproteins, which comprise the protein moiety of lipoproteins,vary in size, density in the aqueous environment of plasma, and lipidand apolipoprotein content. The classification of lipoproteins reflectstheir density in plasma (1.006 gm/mL) as gauged by flotation in theultracentrifuge. For example, triglyceride-rich lipoproteins consistingof chylomicrons (meaning a class of lipoproteins that transport dietarycholesterol and triglycerides after meals from the small intestine totissues for degradation) and very low density lipoprotein (VLDL) have adensity less than 1.06 gm/mL. Id.

Apolipoproteins have four major roles: (1) assembly and secretion of thelipoprotein (apo B100 and B48); (2) structural integrity of thelipoprotein (apo B, apo E, apo A1, apo AII); (3) coactivators orinhibitors of enzymes (apo A1, C1, CII, CIII); and (4) binding ordocking to specific receptors and proteins for cellular uptake of theentire particle or selective uptake of a lipid component (apoA1, B100,E). Id. The role of several apolipoproteins (AIV, AV, D, and J) remainincompletely understood. Id.

Low density lipoprotein (or LDL cholesterol) particles carry cholesterolthroughout the body, delivering it to different organs and tissues. Theexcess keeps circulating in blood. LDL particles contain predominantlycholesteryl esters packaged with the protein moiety apoB100. Id. at1076.

High density lipoproteins (or HDL cholesterol) act as cholesterolscavengers, picking up excess cholesterol in the blood and taking itback to the liver where it is broken down. Apolipoprotein A1, the mainprotein of HDL, is synthesized in the intestine and liver. Lipid-freeApo A1 acquires phospholipids from cell membranes and from redundantphospholipids shed during hydrolysis of triglceride-rich lipoproteins.Lipid-free apo A1 binds to ABCA1 and promotes its phosphorylation viacAMP, which increases the net efflux of phospholipids and cholesterolonto apo A1 to form a nascent HDL particle. Id. These nascent HDLparticles will mediate further cellular cholesterol efflux. Id.

The scavenger receptor class B (SR-B1; also named CLA-1 in humans (Id.,citing Acton, S. et al, “Identification of scavenger receptor SR-B1 as ahigh density lipoprotein receptor,” Science 271: 518 (1996)) and theadenosine triphosphate binding cassette transporter A1 (ABCA1) (Id.,citing Krinbou, L. et al, “Biogenesis and speciation of nascent apoA1-containing particles in various cell lines,” J. Lipid Res. 46: 1668(2005)) bind HDL particles. SR-B1, a receptor for HDL (also for LDL andVLDL, but with less affinity), mediates the selective uptake of HDLcholesteryl esters in steroidogenic tissues, hepatocytes andendothelium. ABCA1 mediates cellular phospholipid (and possiblycholesterol) efflux and is necessary and essential for HDL biogenesis.Id.

Cellular cholesterol homeostasis is achieved via at least four majorroutes: (1) cholesterol de novo biosynthesis from acetyl-CoA in theendoplasmic reticulum; (2) cholesterol uptake by low density lipoprotein(LDL) receptor-mediated endocytosis of LDL-derived cholesterol fromplasma; 3) cholesterol efflux mediated by ABC family transporters suchas ATP-binding cassette, sub-family A (ABC1), member 1(ABCA1)/ATP-binding cassette, sub-family G, member 1 (ABCG1), andsecretion mediated by apolipoprotein B (ApoB); and (4) cholesterolesterification with fatty acids to cholesterol esters (CE) byacyl-coenzyme A:cholesterol acyltransferase (ACAT) (see FIG. 11 (Jiang,W. and Song, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,”Diabetes Metab. 38: 171-80 (2014)).

Cholesterol Biosynthetic Pathways

Cholesterol synthesis takes place in four stages: (1) condensation ofthree acetate units to form a six-carbon intermediate, mevalonate; (2)conversion of mevalonate to activated isoprene units; (3) polymerizationof six 5-carbon isoprene units to form the 30-carbon linear squalene;and (4) cyclization of squalene to form the steroid nucleus, with afurther series of changes to produce cholesterol. (Endo, A., “Ahistorical perspective on the discovery of statins,” Proc. Jpn Acad,Ser. B Phys. Biol. Sci 86(5): 484-93 (2010)).

The mevalonate arm of the cholesterol biosynthesis pathway, whichincludes enzymatic activity in the mitochondria, peroxisome, cytoplasmand endoplasmic reticulum, starts with the consumption of acetyl-CoA,which occurs in parallel in three cell compartments (the mitochondria,cytoplasm, and peroxisome) and terminates with the production ofsqualene in the endoplasmic reticulum (Mazein, A. et al., “Acomprehensive machine-readable view of the mammalian cholesterolbiosynthesis pathway,” Biochemical Pharmacol. 86: 56-66 (2013)). Thefollowing are enzymes of the mevalonate arm:

Acetyl-CoA acetyltransferase (ACAT1; ACAT2; acetoacetyl-CoA thiolase; EC2.3.1.9) catalyzes the reversible condensation of two molecules ofacetylcoA and forms acetoacetyl-CoA. Id.

Hydroxymethylglutaryl-CoA synthase (HMGCS1 (cytoplasmic); HMGCS2(mitochondria and peroxisome); EC 2.3.3.10 catalyzes the formation of3-hydroxy-3-methylglutaryl CoA (3HMG-CoA) from acetyl CoA andacetoacetyl Co A. Id.

Hydroxymethylglutaryl-CoA lysase (mitochondrial, HMGCL; EC 4.1.3.4)transforms HMG-CoA into Acetyl-CoA and acetoacetate (HMGCR; EC 1.1.34)catalyzes the conversion of 3HMG-CoA into mevalonic acid. This step isthe committed step in cholesterol formation. HMGCR is highly regulatedby signaling pathways, including the SREBP pathway.Id.

Mevalonate kinase (MVK; ATP:mevalonate 5-phosphotransferase; EC2.7.1.36) catalyzes conversion of mevalonate into phosphomevalonate. Id.

Phosphomevalonate kinase (PMVK; EC 2.7.4.2) catalyzes formation ofmevalonate 5-diphosphate from mevalonate 5-phosphate. Id.

Diphosphomevalonate decarboxylase (MVD; mevalonate (diphospho)decarboxylase; EC 4.1.1.33) decarboxylates mevalonate 5-diphosphate,forming isopentenyldiphosphate while hydrolyzing ATP. Id.

Isopentenyl-diphosphate delta-isomerases (ID11; ID12; EC 5.3.3.2)isomerize isopentenyl diphosphate into dimethylallyl diphosphate, thefundamental building blocks of isoprenoids. Id.

Farnesyl diphosphate synthase (FDPS; EC2.5.1.10; EC 2.5.1.1;dimethylallyltranstransferase) catalyzes two reactions that lead tofarnesyl diphosphate formation. In the first (EC 2.5.1.1 activity),isopentyl diphosphate and dimethylallyl diphosphate are condensed toform geranyl disphosphate. Next, geranyl diphosphate and isopentenyldiphosphate are condensed to form farnesyl diphosphate (EC 2.5.1.10activity). Id.

Geranylgeranyl pyrophosphate synthase (GGPS1; EC 1.5.1.29; EC 2.5.1.10;farnesyl diphosphate synthase; EC 2.5.1.1;dimethylallyltranstransferase) catalyzes the two reactions of farnesyldiphosphate formation and the addition of three molecules of isopentenyldiphosphate to dimethylallyl diphosphate to form geranylgeranyldiphosphate. Id.

Farnesyl-diphosphate farnesyltransferase 1 (FDFT1; EC 2.5.1.21; squalenesynthase) catalyzes a two-step reductive dimerization of two farnesyldiphosphate molecules (C15) to form squalene (C30). The FDFT1 expressionlevel is regulated by cholesterol status; the human FDFT1 gene has acomplex promoter with multiple binding sites for SREBP-1a and SREBP-2.Id.

The sterols arms of the pathway start with Squalene and terminate withcholesterol production on the Bloch and Kandutsch-Russell pathways andwith 24 (S),25-epoxycholesterol on the shunt pathway. Id. The followingare enzymes of the sterol arms:

Squalene epoxidase (SQLE; EC 1.14.13.132, squalene monooxygenase)catalyzes the conversion of squalene into squalene-2,3-epoxide and theconversion of squalene-2,3-epoxide (2,3-oxidosqualene) into2,3:22,23-diepoxysqualene (2,3:22,23-dioxidosqualene). The firstreaction is the first oxygenation step in the cholesterol biosynthesispathway. The second is the first step in 24(S),25-epoxycholesterolformation from squalene 2,3-epoxide. Id.

Lanosterol synthase (LSS; OLC; OSC; 2,3-oxidosqualene:lanosterolcyclase; EC 5.4.99.7) catalyzes cyclization of squalene-2,3-epoxide tolanosterol and 2,3:22,23-depoxysqualene to 24(S),25-epoxylanosterol. Id.

Delta(24)-sterol reductase (DHCR24; 24-dehydrocholesterol reductase; EC1.3.1.72) catalyzes the reduction of the delta-24 double bond ofintermediate metabolites. In particular it converts lanosterol into 24,25-dihydrolanosterol, the initial metabolite of the Kandutsch-Russelpathway and also provides the last step of the Bloch pathway convertingdesmosterol into cholesterol. Intermediates of the Bloch pathway areconverted by DHCR24 into intermediates of the Kandutsch-Russell pathway.Id.

Lanosterol 14-alpha demethylase (CYP51A1; cytochrome P450, family 51,subfamily A, polypeptide 1; EC 1.14.13.70) converts lanosterol into4,4-dimethyl-5α-cholesta-8,14,24-trien-3β-ol and 24,25-dihydrolanosterolinto 4,4-dimethyl-5α-cholesta-8,14-dien-3β-ol in three steps. Id.

Delta (14)-sterol reductase (TM7F2; transmembrane 7 superfamily member2, EC 1.3.1.70) catalyzes reactions on the three branches of thecholesterol and 24(S),25-epoxycholesterol pathways. Id.

Methylsterol monooxygenase 1 (MSM01; SC4MOL; C-4 methylsterol oxidase;EC 1.14.13.72) catalyzes demethylation of C4 methylsterols. Id.

Sterol-4-alpha-carboxylate 3-dehydrogenase, decarboxylating (NSDHL;NAD(P) dependent steroid dehydrogenase-like; EC 1.1.1.170) participatesin several steps of post-squalene cholesterol and24(S),25-epoxycholeseterol synthesis. Id.

3-keto-steroid reductase (HSD17B7; 17-beta-hydroxysteroid dehydrogenase7; EC 1.1.1.270) converts zymosterone into zymosterol in the Blochpathway. Id.

3-Beta-hydroxysteroid-delta(8),delta(7)-isomerase (EBP; emopamil-bindingprotein; EC5.3.3.5) catalyzes the conversion of delta(8)-sterols intodelta(7)-sterols. Id.

Lathosterol oxidase (SC5DL; sterol-C5-desaturase (ERG3delta-5-desaturase homolog, S. cerevisiae-like; EC 1.14.21.6) catalyzesthe production of 7-dehydrocholesterol, 7-dehydrodesmosterol and24(S),25-epoxy-7-dehydrocholesterol. Id.

7-dehydrocholesterol reductase (DHCR7; EC 1.3.1.21) catalyzes reductionof the C7-C8 double bond of 7-dehydrocholesterol and formation ofcholesterol, and produces desmosterol from 7-dehydrodesmosterol and24(S),25-epoxycholesterol from 24(S),25-epoxy-7-dehydrocholesterol. Id.

Cytochrome P450, family 3, subfamily A, polypeptide 4 (CYP3A4;1,8-cineole 2-exo-monooxygenase; taurochenodeoxycholate 6α-hydroxylase;EC 1.14.13.97)) catalyzes the hydroxylation of cholesterol leading to25-hydroxycholesterol and 4β-hydroxycholesterol. Id.

Cholesterol 25-hydroxylase (CH25H; cholesterol 25-monooxygenase; EC1.14.99.38) uses di-iron cofactors to catalyze the hydroxylation ofcholesterol to produce 25-hydroxycholesterol, and has the capacity tocatalyze the transition of 24-hydroxycholesterol to 24,25-dihydroxycholesterol. Id.

Cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1;cholesterol 7-alpha-hydroxylase; EC 1.14.13.17) is responsible forintroducing a hydrophilic moiety at position 7 of cholesterol to form7α-hydroxycholesterol. Id.

Cytochrome P450, family 27, subfamily A, polypeptide 1 (CYP27A1; Sterol27-hydroxylase; EC 1.14.13.15) catalyzes the transition of mitochondrialcholesterol to 27-hydroxycholesterol and 25-hydroxycholesterol. Id.

Cytochrome P450 46A1 (CYP46A1, cholesterol 24-hydroxylase, EC1.14.13.98) catalyzes transformation of cholesterol into24(S)-hydroxycholesterol. Id.

Intermediates in Cholesterol Synthesis as Physiological Regulators

Intermediates in cholesterol synthesis, mostly sterols (e.g.,7-dehydrocholesterol, which is converted to cholesterol by DHCR7(7-dehydrocholesterol reductase), but which also is a precursor forvitamin D), have been credited with having regulatory functions distinctfrom those of cholesterol. (Sharpe, L J and Brown, A J, “Controllingcholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase(HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)).

C4-methylsterols are produced by lanosterol 14α-demethylase (encoded byCYP51A1 (cytochrome P450, family 51, subfamily A, polypeptide 1) anddemethylated by SC4MOL (sterol-C4-methyl oxidase like 1; methylsterolmonooxygenase 1) and its partner, NSDHL (NAD(P)-dependent steroiddehydrogenase-like; sterol-4-α-carboxylate 3-dehydrogenase,decarboxylating),Id.

24, 25-dihydrolanosterol purportedly is the primary degradation signalfor 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) (Id., citing Song,B L, et al, “Insig-mediated degradation of HMG-CoA reductase stimulatedby lanosterol, an intermediate in the synthesis of choleseterol,” CellMeta. 1: 179-89 (2005); Lange, Y. et al, “Effectors of rapid homeostaticresponses of endoplasmic reticulum cholesterol and3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Chem. 283: 1445-55(2008)).

The nonsterol intermediate squalene has been implicated in stimulatingHMGCR degradation (Id., citing Leichner, G S, et al, “Metabolicallyregulated endoplasmic reticulum-associated degradation of3-hydroxy-3-methylglutaryl-CoA reductase. Evidence for requirement of ageranylgeranylated protein,” J. Biol. Chem. 286: 32150-61 (2011)).

A number of cholesterol synthesis intermediates can serve as activatingligands of the nuclear liver X receptor (LXR), which up-regulatescholesterol export genes and represses inflammatory genes. These sterolsinclude 24,25-dihydrolanosterol (Id., citing Zhu, J. et al, “Effects ofFoxO4 overexpression on cholesterol biosynthesis, triacylglycerolaccumulation, and glucose uptake,” J. Lipid Res. 51: 1312-24 (2010)),meiosis-activating sterols (MASs) (Id., citing He, M, et al, “Mutationsin the human SC4MOL gene encoding a methyl sterol oxidase causepsoriasiform dermatitis, microcephaly, and developmental delay,” J.Clin. Invest. 121: 97 6-984 (2011)) and desmosterol (Id., citing Yang,C. et al, “Sterol intermediates from choleseterol biosynthetic pathwayas liver X receptor ligands,” J. Biol. Chem. 281: 27816-826 (2006);Spann, N J et al, “Regulated accumulation of desmosterol integratesmacrophage lipid metabolism and inflammatory responses,” Cell 151:138-52 (2012)).

The oxysterol 24(S),25-epoxycholesterol (24,25-EC), a potent LXR agonist(Id., citing Lehmann, J M et al, “Activation of the nuclear receptor LXRby oxysterols defines a new hormone response pathway,” J. Biol. Chem.272: 3137-40 (1997)), is produced in a shunt pathway in sterol synthesis(Id., citing Spencer, T A, et al, “24(S),25-epoxyscholesterol. Evidenceconsistent with a role in the regulation of hepatic cholestrogenesis,”J. Biol. Chem. 260: 13391-94 (1985)), and its production is determinedby the relative activities of squalene monooxygenase (SM) and lanosterolsynthase (LS). Partial inhibition or knockdown of LS diverts more fluxinto the shunt pathway, producing more 14,15-epoxycholesterol (14,15-EC)(Id., citing Dang, H. et al, “Suppression of 2,3-oxidosqualene cyclaseby high fat diet contributes to liver X receptor-α-mediated improvementof hepatic lipid profile,” J. Biol. Chem. 284: 6218-26 (2009)), whereasoverexpression of LS abolishes 24,25-EC production (Id., citing Wong, J.et al, “Endogenous 24(S),25-epoxycholesterol fine-tunes acute control ofcellular cholesterol homeostasis,” J. Biol. Chem. 283: 700-707 (2008)).Conversely, overexpression of SM increases 24,25-EC production (Id.,citing Zerenturk, E J et al, “The endogenous regulator24(S),25-epoxycholesterol inhibits choleseterol synthesis at DHCR24(Seladin-1),” Biochim. Biophys. Acta 1821: 1269-77 (2012)). The extentto which SM and LS are differentially regulated to alter 14,15-ECproduction is not known.

Cholesterol Uptake by Low Density Lipoprotein (LDL) Receptor-MediatedEndocytosis of LDL-Derived Cholesterol from Plasma

The LDL receptor regulates the entry of cholesterol into cells; tightcontrol mechanisms alter its expression on the cell surface, dependingon need. Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D.Zipes, Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at1072. Other receptors for lipoproteins include several that bind VLDL,but not LDL. Id. The LDL receptor-related peptide, which mediates theuptake of chylomicron remnants and VLDL, preferentially recognizesapolipoprotein E (apo E) (Id., citing Hiltunen, T P et al, Expression ofLDL receptor, VLDL receptor, LDL receptor-related protein, and scavengerreceptor in rabbit atherosclerotic lesions: Marked induction ofscavenger receptor and VLDL receptor expression during lesiondevelopment,” Circulation 97: 1079 (1998)). The LDL receptor-relatedpeptide interacts with hepatic lipase. A specific VLDL receptor alsoexists (Id., citing Nimph, J, and Schneider, W J, “The VLDL receptor: anLDL receptor relative with eight ligand binding repeats, LR8.Atherosclerosis 141: 191-202 (1998)). The interaction betweenhepatocytes and the various lipoproteins containing apo E is complex andinvolves cell surface proteoglycans that provide a scaffolding forlipolytic enzymes (lipoprotein lipase and hepatic lipase) involved inremnant lipoprotein recognition (Id., citing Mahley, R W, Ji, Zs,“Remnant lipoprotein metabolism: key pathways involving cell-surfaceheparin sulfate proteoglycans and apolipoprotein E,” J. Lipid Res. 40:1-(1999); Barown M I et al, “A macrophage receptor for apolipoproteinB48: cloning, expression and atherosclerosis, Proc. Natl Acad. Sci. USA97: 7488 (2000); de Man, F H et al, “Lipolysis of very low densitylipoproteins by heparin sulfate proteoglycan-bound lipoprotein lipase,”J. Lipid Res. 38: 2465 (1997)).

Macrophages express receptors that bind modified (especially oxidized)lipoproteins. These scavenger lipoprotein receptors mediate the uptakeof oxidized LDL into macrophages. In contrast to the regulated LDLreceptor, high cellular cholesterol content does not suppress scavengerreceptors, enabling the intimal macrophages to accumulate abundantcholesterol, become foam cells, and form fatty streaks. Endothelialcells also can take up modified lipoproteins through a specificreceptor, such as Lox-1 (Sawamura, T. et al, “an endothelial receptorfor oxidized low-density lipoprotein,” Nature 386: 73 (1997)).

Cholesterol Efflux is Mediated by ABC Family Transporters Such asATP-Binding Cassette, Sub-Family A (ABC1), Member 1 (ABCA1)/ATP-BindingCassette, Sub-Family G, Member 1 (ABCG1), and Secretion Mediated byApolipoprotein B (ApoB);

Because most cells in the body do not express pathways for catabolizingcholesterol, efflux of cholesterol is critical for maintaininghomeostasis. (Phillips, M C, “Molecular Mechanisms of CellularCholesterol Efflux,” J. Biol. Chem. 289 (35): 24020-29 (2014)). Highdensity lipoprotein (HDL) comprises a heterogeneous population ofmicroemulsion particles 7-12 nm in diameter containing a core ofcholesterol ester (CE) and triglyceride (TG) molecules stabilized by amonomolecular layer of phospholipid (PL) and apolipoprotein (apo), ofwhich apo1 is the principal component (Id. citing Phillips, M C, “Newinsights into the determination of HDL structure by apolipoproteins,” J.Lipid Res. 54: 2034-48 (2013)). The presence of PL in the particlesenables HDL to solubilize and transport unesterified (free) cholesterol(FC) released from cells, thereby mediating removal of cholesterol fromcholesterol-loaded arterial macrophages and transport to the liver forcatabolism and elimination from the body (“reverse cholesteroltransport”) (Id., citing Rothblat, G H and Phillips, M C, “High-densitylipoprotein heterogeneity and function in reverse cholesteroltransport,” Curr. Opin. Lipidol. 21: 229-38 (2010); Rosenson, R S et al,“Cholesterol efflux and atheroprotection: advancing the concept ofreverse cholesterol transport,” Circulation 125: 1905-19 (2012)).

The first step in reverse cholesterol transport is efflux of FC from thecell plasma membrane to HDL. Id. In the case of macrophages, four effluxpathways have been identified: the aqueous diffusion efflux pathway, thescavenger receptor class B, type 1 (SR-B1) pathway; the ATP bindingcassette transporter G1 (ABCG1) pathway and the ATP-binding cassettetransporter A1 (ABCA1) pathway. Id. The first two processes, which arepassive, involve simple diffusion (aqueous diffusion pathway) andfacilitated diffusion (SR-B1-mediated pathway). Id. The two activeprocesses involve members of the ATP-binding cassette (ABC) family oftransmembrane transporters, namely ABCA1 and ABCG1. Id. The efficiencyof an individual serum sample in accepting cellular cholesterol dependsupon both the distribution of HDL particles present and the levels ofcholesterol transporters expressed in the donor cells. Id.

Aqueous Diffusion Efflux Pathway

HDL is the component of serum responsible for mediating FC efflux frommonolayers of mouse L-cell fibroblasts. Id. Transfer occurs by anaqueous phase intermediate where monomeric FC molecules desorb from adonor particle and diffuse until they are absorbed by an acceptorparticle. The rate of transfer of the highly hydrophobic cholesterolmolecule from donor to acceptor is limited by the rate of desorptioninto the aqueous phase, which is sensitive to the physical state of thephospholipid (PL) milieu in which the transferring FC molecules arelocated. The net mass FC efflux from cells to HDL in the extracellularmedium is promoted by metabolic trapping in which return of released FCto the cell is prevented by esterification, when lecithin-cholesterolaceyltransferase acts on HDL (Id., citing Czarnecka, H. and Yokoyama,S., “Regulation of cellular cholesterol efflux by lecithin: cholesterolacyltransferase reaction through nonspecific lipid exchange,” J. Biol.Chem. 271: 1023-27 (1996)).

SR-B1 Efflux Pathway

SR-B1 is a member of the CD36 superfamily of scavenger receptor proteinsthat also includes lysosomal integral membrane protein-2 (LIMP-2). Id.The receptor is most abundantly expressed in liver, where it functionsin the reverse cholesterol transport pathway and in steroidogenictissue, where it mediates cholesterol delivery (Id., citing Zannis, V.et al, “Role of apoA-1, ABCA1, LCAT and SR-B1 in the biogenesis of HDL,”J. Mol. Med. 84: 276-94 (2006)). It is a homo-oligomeric glycoproteinlocated in the plasma membrane with two N- and C-terminal transmembranedomains and a large central extracellular domain (Id., citing Williams,D L, et al, “Scavenger receptor B1 and cholesterol trafficking,” Curr.Opin. Lipidol. 10: 329-39 (1999); Meyer, J M et al, “New developments inselective cholesteryl ester uptake,” Curr. Opin. Lipidol. 24: 386-92(2013)). In 1996, it was established that SR-B1 is an HDL receptor thatmediates cholesterol uptake into cells. This process involves selectivetransfer of the cholesterol ester (CE) in an HDL particle into the cellwithout endocytic uptake and degradation of the HDL particle itself. Inaddition to promoting delivery of HDL cholesterol to cells, SR-B1 alsoenhances efflux of cellular cholesterol to HDL (Id., citing Ji, Y et al,“Scavenger receptor B1 promotes high density lipoprotein-mediatedcellular cholesterol efflux,” J. Biol. Chem. 272: 20982-985 (1997);Jian, B. et al, “Scavenger receptor class B type 1 as a mediator ofcellular cholesterol efflux to lipoproteins and phospholipid acceptors,”J. Biol. Chem. 273: 5599-5606 (1998)) with the two processes beingrelated (Id., citing Gu, X et al, “Scavenger receptor class B, type1-mediated [3H]cholesterol efflux to high and alow density lipoproteinsis dependent on lipoprotein binding to the receptor,” J. Biol. Chem.275: 29993-30001 (2000)). For CE selective uptake via SR-B1, HDL bindingand CE uptake are tightly coupled. The mechanism for CE uptake from HDLinvolves a two-step process in which HDL first binds to the receptor andthen CE molecules transfer from the bound HDL particle into the cellplasma membrane, with enhanced binding of larger HDL particles to SR-B1increasing the selective delivery of CE (Id., citing Thuahnai, S T, etal, “SR-B1-mediated cholesteryl ester selective uptake and efflux ofunesterified cholesterol: influence of HDL size and structure,”” J.Biol. Chem. 279: 12448-455 (2004)). The binding of HDL to theextracellular domain of SR-B1 involves direct protein-protein contactwith a recognition motif being the amphipathic α helix characteristic ofHDL apolipoproteins (Id., citing Williams, D L et al, “Binding andcross-linking studies show that scavenger receptor B1 interacts withmultiple sites in apolipoprotein A-1 and identify the class Aamphipathic α helix as a recognition motif,” J. Biol. Chem. 275:18897-18904 (2000). Consistent with CE selective uptake being a passiveprocess, the rate of uptake is proportional to the amount of CEinitially present in the HDL particles.

FC efflux and HDL binding are not completely coupled, and the FC effluxmechanism proceeds by different pathways at low and high extracellularHDL concentrations (Id., citing Thuahnai, S T, et al, “SR-B1-mediatedcholesteryl ester selective uptake and efflux of unesterifiedcholesterol: influence of HDL size and structure,”” J. Biol. Chem. 279:12448-455 (2004); de la Llera-Moya, M. et al, “Scavenger receptor B1(SR-B1) mediates free cholesterol flux independently of HDL tethering tothe cell surface,” J. Lipid Res. 40: 575-80 (1999)). At low HDLconcentrations, binding of HDL to SR-B1 is critical, allowingbidirectional FC transit through the hydrophobic tunnel present in theextracellular domain of the receptor. Because the FC concentrationgradient between the bound HDL particle and the cell plasma membrane isopposite to that of CE, the relatively high FC/PL ratio in the plasmamembrane causes the direction of net mass FC transport to be out of thecell. Consistent with this concept, enhancing the PL content of HDLpromotes FC efflux from cells (Id., citing Yancey, P G, et al, “Highdensity lipoprotein phospholipid composition is a major determinant ofthe bi-directional flux and net movement of cellular free cholesterolmediated by scavenger receptor B1,” J. Biol. Chem. 275: 36596-36604(2000)). Larger HDL particles promote more FC efflux than smaller HDL,because they bind better to SR-B1 (Id., citing Thuahnai, S T, et al,“SR-B1-mediated cholesteryl ester selective uptake and efflux ofunesterified cholesterol: influence of HDL size and structure,”” J.Biol. Chem. 279: 12448-455 (2004)). At higher HDL concentrations wherebinding to the receptor is saturated, FC efflux still increases withincreasing HDL concentration (Id., citing Thuahnai, S T, et al,“SR-B1-mediated cholesteryl ester selective uptake and efflux ofunesterified cholesterol: influence of HDL size and structure,”” J.Biol. Chem. 279: 12448-455 (2004)), because SR-B1 induces reorganizationof the FC in the cell plasma membrane.

ABCG1 Efflux Pathway

ABCG1 functions as a homodimer, and is expressed in several types, whereit mediates cholesterol transport through its ability to translocatecholesterol and oxysterols across membranes. Id. Expression of ABCG1enhances FC and PL efflux to HDL (Id., citing Wang, N. et al,“ATP-binding cassette transporters G1 and G4 mediate cellularcholesterol efflux to high-density lipoproteins,” Proc. Natl Acad. Sci.USA 101: 9774-79 (2004); Kennedy, M A et al, “ABCG1 has a critical rolein mediating cholesterol efflux to HDL and preventing cellular lipidaccumulation,” Cell Metab. 1: 121-31 (2005)), but not to lipid-freeapoA-1 (Id., citing Vaughan, A M and Oram, J F, “ABCG1 redistributescell cholesterol to domains removable by high density lipoprotein butnot by lipid-depleted apolipoproteins,” J. Biol. Chem. 280: 20150-57(2005); Sankaranarayanan, S. et al., “Effects of acceptor compositionand mechanism of ABCG1-mediated cellular free cholesterol efflux,” J.Lipid Res. 50: 275-84 (2009)). The presence of the transporter inducesreorganization of plasma membrane cholesterol so that it becomesaccessible to cholesterol oxidase (Id., citing Vaughan, A M and Oram, JF, “ABCG1 redistributes cell cholesterol to domains removable by highdensity lipoprotein but not by lipid-depleted apolipoproteins,” J. Biol.Chem. 280: 20150-57 (2005)), creating an activated pool of plasmamembrane FC, and desorption of FC molecules from this environment intothe extracellular medium is facilitated. Increased expression of ABCG1enhances FC efflux to HDL2 and HDL3 similarly, but has no effect on theinflux of FC from these lipoprotein particles.

ABCA1 Efflux Pathway

ABCA1 is a full transporter whose expression is up-regulated bycholesterol loading, which leads to enhanced FC efflux. Id. Binding andhydrolysis of ATP by the two cytoplasmic, nucleotide-binding domainscontrol the conformation of the transmembrane domains so that theextrusion pocket is available to translocate substrate from thecytoplasmic leaflet to the exofacial leaflet of the bilayer membrane.Id. ABCA1 actively transports phosphatidylcholine, phosphatidylserine,and sphingomyelin with a preference for phosphatidylcholine (Id., citingQuazi, F and Molday, R S, “Differential phospholipid substrates anddirectional transport by ATP-binding cassette proteins ABCA, ABCA7, andABCA4 and disease-causing mutants,” J. Biol. Chem. 288: 34414-26(2013)). This PL translocase activity leads to the simultaneous effluxof PL and FC (Id., citing Gillotte, K L, et al, “Removal of cellularcholesterol by pre-β-HDL involves plasma membrane microsolubilization,”J. Lipid Res. 39: 1918-28 (1998); Smith, J D et al, “ABCA1 mediatesconcurrent cholesterol and phospholipid efflux to apolipoprotein A-1,”J. Lipid Res. 45: 635-44 (2004)) to lipid-free apoA-1 (plasmapre-β1-HDL). The cellular FC released to apoA-1 originates from both theplasma membrane and the endosomal compartment (Id., citing Chen, W. etal, “Preferential ATP-binding cassette transporter A1-mediatedcholesterol efflux from late endosomes/lysosomes,” J. Biol. Chem. 276:43564-69 (2001)).

The PL translocase activity of ABCA1 induces reorganization of lipiddomains in the plasma membrane (Id., citing Landry, Y D, et al,“ATP-binding cassette transporter A1 expression disruptsraft membranemicrodomains through its ATPase-related functions,” J. Biol. Chem. 281:36091-101 (2006)). ABCA1 exports PL and FC to various plasmaapolipoproteins. Besides FC efflux, intracellular signaling pathways areactivated by the interaction of apoA-1 with ABCA1 (Id., citing Mineo, C.and Shaul, P W, “Regulation of signal transduction by HDL,” J. LipidRes. 54: 2315-24 (2013); Liu, Y, and Tang, C., “Regulation of ABCA1functions by signaling pathways,” Biochim. Biophys. Acta, 1821: 522-29(2012)).

It is well established that the activity of ABCA1 in the plasma membraneenhances binding of apoA-1 to the cell surface, but there has beencontroversy about the role of this binding in the acquisition ofmembrane PL by apo-A1. Id. It has been proposed that apoA-1 acquires PLeither directly from ABCA1 while it is bound to the transporter, orindirectly at a membrane lipid-binding site created by ABCA1 activity.Id.

The ABCA1-mediated assembly of nascent HDL particles occurs primarily atthe cell surface (Id., citing Faulkner, L E, et al, “An analysis of therole of a retroendocytosis pathway in ABCA1-mediated cholesterol effluxfrom macrophages,” J. Lipid Res. 49: 1322-32 (2008); Denis, M. et al,“ATP-binding cassette A-1-mediated lipidation of apolipoprotein A-1occurs at the plasma membrane and not in the endocytic compartments,” J.Biol. Chem. 283: 16178-186 (2008)), where extracellular apoA-1 for HDLparticle formation is available. The FC/PL ratio in nascent HDLparticles created by ABCA1 activity is dependent upon the cell type andmetabolic status of the cell, but the population of larger particles isalways relatively FC-rich as compared with the smaller particles.

Regulation of cholesterol efflux depends in part on the ABCA1 pathway,controlled in turn by hydroxysterols (especially 24 and 27-OHcholesterol, which act as ligands for the liver-specific receptor (LXR)family of transcriptional regulatory factors. Braunwald's Heart Disease,P. Libby, R. Bonow, D. Mann and D. Zipes, Eds., 8^(th) Edition, SaundersElsevier, Philadelphia, Pa. (2008) at 1076.

It has been demonstrated by in vivo genetic evidence that ABCA1 effluxfunction has anti-cancer activity, which is compromised followinginhibition of ABCA1 gene expression by oncogenic mutations orcancer-specific ABCA1 loss-of-function mutations. Smith, B. and Land,H., “Anticancer activity of the cholesterol exporter ABCA1 gene,” CellRep. 22(3): 580-90 (2012), It has been suggested that loss ofABCA1-mediated efflux is a key step in allowing the accumulation ofmitochondrial cholesterol levels that support cancer survival. Id. Theyinterpreted their data as supporting a causal role for elevatedmitochondrial cholesterol in cancer cells, and hypothesized that inconcert with elevated cholesterol synthesis found in cancer cells,deficiency of ABCA1 allows for increased mitochondrial cholesterol,inhibits release of mitochondrial cell death-promoting molecules, andthus facilitates cancer cell survival. Id.

Cholesterol Esterification with Fatty Acids to Cholesterol Esters (CE)by Acyl-Coenzyme A:Cholesterol Acyltransferase (ACAT)

Cholesterol content in membranes regulates the cholesterolacyltransferase (CAT) pathway at the level of protein regulation.(Braunwald's Heart Disease, P. Libby, R. Bonow, D. Mann and D. Zipes,Eds., 8th Edition, Saunders Elsevier, Philadelphia, Pa. (2008) at 1076,citing Willner, E. et al, “Deficiency of acyl CoA:cholesterolaceyltransferase 2 prevents atherosclerosis in apolipoproteinE-deficient mice., Proc. Natl Acad. Sci. USA 100: 1262 (2003). Humansexpress two separate forms of ACAT (ACT1 and ACAT2), which derive fromdifferent genes and mediate cholesterol esterification in cytoplasm andin the endoplasmic reticulum lumen for lipoprotein assembly andsecretion.

Regulation of Cholesterol Content

Under conditions of cell cholesterol sufficiency, the cell can decreaseits input of cholesterol by decreasing the de novo synthesis ofcholesterol. The cell can also decrease the amount of cholesterol thatenters the cell via the LDL-R, increase the amount stored as cholesterylesters, and promote the removal of cholesterol by increasing itsmovement to the plasma membrane for efflux.

The regulation of HMG CoA reductase, the rate limiting step incholesterol biosynthesis, has been investigated in detail. However, thisenzyme acts very early in the cholesterol synthesis pathway. There isaccumulating evidence that enzymes beyond HMG CoA reductase serve asflux controlling points, and that regulation of cholesterol synthesiscan occur at multiple levels throughout the pathway. (Sharpe, L J andBrown, A J, “Controlling cholesterol synthesis beyond3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J. Biol. Chem. 288(26): 18707-715 (2013)).

Transcriptional Regulation

Sterol Regulatory Element-Binding Proteins (SREBPs)

SREBPs, membrane bound transcription factors that coordinate thesynthesis of fatty acids and cholesterol, the two major building blocksof membranes (Brown, M S & Goldstein, J L, “The SREBP pathway:regulation of cholesterol metabolism by proteolysis of a membrane-boundtranscription factor,” Cell 89: 331-40 (1997)), belong to the basichelix-loop-helix-leucine zipper (bHLH-Zip) family of transcriptionfactors. There are three SREBP proteins (SREB-1a, SREBP-1c, and SREBP-2)from two srebp genes designated srebp1 and srebp2. Id. The SREBP2isoform plays a major role in regulating cholesterol synthetic genes.

As shown in Table 1, nearly all of the genes encoding cholesterolsynthesis enzymes are SREBP targets.

Gene Name Gene Symbol SREBP Target Acetyl-CoA acetyltransferase, ACAT2Yes cytosolic 3-hydroxy-3-methylglutaryl- MHGCS1 Yes CoA synthase 1(soluble) 3-hydroxy-3-methylglutaryl- HMGCR Yes CoA reductase Mevalonatekinase MVK Yes Phosphomevalonate kinase PMVK Yes Mevalonate MVD Yes(diphospho)decarboxylase Isopentenyl-diphosphate Δ- ID11/ID12 Yesisomerase ½ Farnesyl-diphosphate FDFS Yes synthaseGeranylgeranyl-diphosphate GGPS1 Yes synthase 1 Farnesyl-diphosphateFDFT1 Yes farnesyltransferase 1 Squalene epoxidase SQLE Yes Lanosterolsynthase (2,3- LSS Yes oxidosqualene-lanosterol cyclase) CytochromeP450, family 51, CYPS1A1 Yes subfamily A, polypeptide 1 Transmembrane 7TM75F2 Yes superfamily member 2 Lamin B receptor LBR No Methylsterolmonooxygenase 1 SCAMOL Yes NAD(P)-dependent steroid NSDHL Yesdehydrogenase-like Hydroxysteroid 17β- HSD17B7 Yes dehydrogenase 7Emopamil-binding protein EBP Yes (sterol isomerase) Sterol C5-desaturaseSC5D Yes 7-Dehydrocholesterol DHCR7 Yes reductase 24 DehydrocholesterolDHCR24 Yes reductaseTaken from Sharpe, L J and Brown, A J, “Controlling cholesterolsynthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J.Biol. Chem. 288 (26): 18707-715 (2013))

SREBPs coordinately regulate the cholesterol biosynthetic pathway andreceptor-mediated endocytosis of LDL at the level of gene transcription.(Brown, M S and Goldstein, J L, “A proteolytic pathway that controls thecholesterol content of membranes, cells and blood,” Proc. Natl Acad.Sci. USA 96: 11041-48 (1999)). In the cholesterol biosynthetic pathway,SREBPs regulate transcription of HMG CoA reductase as well astranscription of genes encoding many other enzymes in the cholesterolbiosynthetic pathway, including HMG CoA synthase, farnesyl diphosphatesynthase and squalene synthase. Id. Studies investigating regulation ofthe DHCR24 promoter provided evidence of binding sites for SREBP-2[Daimiel, L A, et al, “Promoter analysis of the DHCR24 (3β-hydroxysterol424-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding-protein)-mediated activation,” Biosci. Rep.(2013)/art:e000/doi 10.1042/BSR20120095); Zerenturk, E J, et al,“Sterols regulate 3β-hydroxysterolΔ24-reductase (DHCR24) via dual sterolregulatory elements: cooperative induction of key enzymes in lipidsynthesis by sterol regulatory element binding proteins,” Biochim. EtBiophys. Acta 1821 (10): 1350-60 (2012)). The SREBPs also regulate theLDL receptor, which supplies cholesterol through receptor mediatedendocytosis, and modulate transcription of genes encoding enzymes offatty acid synthesis and uptake, including acetyl CoA carboxylase, fattyacid synthase, stearoyl CoA desaturase-1 and lipoprotein lipase. (Brown,M S & Goldstein, J L, “The SREBP pathway: regulation of cholesterolmetabolism by proteolysis of a membrane-bound transcription factor,”Cell 89: 331-40 (1997)).

Nascent SREBPs are targeted to the endoplasmic reticulum (ER) membranewithout any transcription activity, because they are not available fortheir target genes, which are located in the nucleus. (Brown, M S &Goldstein, J L, “The SREBP pathway: regulation of cholesterol metabolismby proteolysis of a membrane-bound transcription factor,” Cell 89:331-40 (1997)). To enhance transcription when cellular sterol is low,the active NH₂-terminal domains of SREBPs are released from endoplasmicreticulum membranes by two sequential cleavages that must occur in theproper order. The first is catalyzed by Site-1 protease (S1P), amembrane bound subtilisin-related serine protease that cleaves thehydrophilic loop of SREBP that projects into the endoplasmic reticulumlumen. (Brown, M S and Goldstein, J L, “A proteolytic pathway thatcontrols the cholesterol content of membranes, cells and blood,” Proc.Natl Acad. Sci. USA 96: 11041-48 (1999)). The second cleavage, atSite-2, requires the action of S2P, a hydrophobic protein that appearsto be a zinc metalloprotease, and takes place within a membrane-spanningdomain of SREBP. Id. Sterols block SREBP processing by inhibiting S1P.Id. Sterols block the proteolytic release process by selectivelyinhibiting cleavage by S1P; S2P is regulated indirectly because itcannot act until SREBP has been processed by S1P. Id.

SREBP cleavage-activating protein (SCAP), an integral ER membraneregulatory protein, is required for cleavage at Site 1 and is the targetfor sterol suppression of this cleavage, i.e., SCAP loses its activitywhen sterols overaccumulate in cells. Id. Within cells, SCAP is found ina tight complex with SREBPs. Id. SCAP contains two distinct domains: ahydrophobic N-terminal domain that spans the membrane eight times and ahydrophilic C-terminal domain that projects into the cytosol.(DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis:Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,”Cell Res. 18 (6): 609-21 (2008)) A 160 amino acid segment of themembrane domain of SCAP has been termed the sterol-sensing domain.(Brown, M S and Goldstein, J L, “A proteolytic pathway that controls thecholesterol content of membranes, cells and blood,” Proc. Natl Acad.Sci. USA 96: 11041-48 (1999)). The C-terminal domain of SCAP mediates aconstitutive association with SREBPs, which is required forSCAP-dependent translocation of SREBPs from the ER to Golgi insterol-deprived cells. (DeBose-Boyd, R. A. “Feedback Regulation ofCholesterol Synthesis: Sterol-accelerated ubiquitination and degradationof HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008)). TheNH₂-terminal bHL-Zip domain with full transcription activity is releasedfrom the membrane to reach the nucleus and act as a transcription factorto activate genes responsible for cholesterol and fatty acidbiosynthesis and LDL uptake (Brown, M S and Goldstein, J L, “Aproteolytic pathway that controls the cholesterol content of membranes,cells and blood,” Proc. Natl Acad. Sci. USA 96: 11041-48 (1999)).

When sterols build up within cells, the proteolytic release of SREBPsfrom ER membranes is blocked, the NH₂-terminal domains that have alreadyentered the nucleus are rapidly degraded, and, as a result,transcription of all of the target genes declines. (Id). This decline iscomplete for the cholesterol biosynthetic enzymes whose transcription isentirely dependent on SREBPs, but less complete for the fatty acidbiosynthetic enzymes whose basal transcription can be maintained byother factors.

Other Factors

Besides SREBP, numerous other transcription factors have been implicatedin the transcriptional control of the various enzymes in cholesterolbiosynthesis. (Sharpe, L J and Brown, A J, “Controlling cholesterolsynthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J.Biol. Chem. 288 (26): 18707-715 (2013)).

Liver X Receptors (LXRs)

Liver X receptors (LXRs) are ligand-activated transcription factors ofthe nuclear receptor superfamily. (Baranowski, M., “Biological role ofliver X receptors,” J. Physiol. Pharmacology. 59 Suppl. 7: 31-55(2008)). There are two LXR isoforms (termed alpha and beta), which, uponactivation, form heterodimers with retinoid X receptor and bind to LXRresponse elements found in the promoter region of the target genes. Id.High expression levels of LXRα in metabolically active tissues fit witha central role of the receptor in lipid metabolism, while LXRβ is moreubiquitously expressed. (Pehkonen, P. et al., “Genome-wide landscape ofliver X receptor chromatin binding and gene regulation in humanmacrophages,” BMC Genomics 13: 50 (2012)). Both LXRs are found invarious cells of the immune system, such as macrophages, dendritic cellsand lymphocytes. Id. In macrophages, the accumulation of excesslipoprotein-derived cholesterol activates LXR and triggers the inductionof a transcriptional program for cholesterol efflux, such as ATP-bindingcassette transporter (ABC) A1 (ABCA1) and ABCG1, while in parallel thereceptor transrepresses inflammatory genes, such as inducible nitricoxide synthase, interleukin 1β, and monocyte chemotactic protein-1. Id.LXR has been reported to regulate cholesterol biosynthesis by directlysilencing the gene expression of two cholesterogenic enzymes (FDFT1 andCYP51A1). (Sharpe, L J and Brown, A J, “Controlling cholesterolsynthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR),” J.Biol. Chem. 288 (26): 18707-715 (2013), citing Wang, Y. et al,“Regulation of cholesterologenesis by the oxysterol receptor, LXRα,” J.Biol. Chem. 283: 26332-339 (2008)).

Endogenous agonists of the LXRs include oxysterols, which are oxidizedcholesterol derivatives. (Baranowski, M., “Biological role of liver Xreceptors,” J. Physiol. Pharmacol. 59 Suppl. 7: 31-55 (2008)). LXRs havebeen characterized as key transcriptional regulators of lipid andcarbohydrate metabolism, and were shown to function as sterol sensorsprotecting the cells from cholesterol overload by stimulating reversecholesterol transport and activating its conversion to bile acids in theliver. Id. This finding led to identification of LXR agonists as potentanti-atherogenic agents in rodent models of atherosclerosis. Id.However, first-generation LXR activators were also shown to stimulatelipogenesis via SREBP1c leading to liver steatosis andhypertriglyceridemia. Id.

Despite their lipogenic action, LXR agonists possess antidiabeticproperties. Id. LXR activation normalizes glycemia and improves insulinsensitivity in rodent models of type 2 diabetes and insulin resistance.Id. Although antidiabetic action of LXR agonists is thought to resultpredominantly from suppression of hepatic gluconeogenesis, some studiessuggest that LXR activation may also enhance peripheral glucose uptake.Id.

Published reports of anti-proliferative effects of synthetic LXR ligandson breast, prostate, ovarian, lung, skin, and colorectal cancer cellssuggest that LXRs are potential targets in cancer prevention andtreatment. Nguyen-Vu, T. et al, “Liver x receptor ligands disrupt breastcancer cell proliferation through an E2F-mediated mechanism,” BreastCancer Res. 15: R51 (2013). Cell line-specific transcriptional responsesand a set of common responsive genes were shown by microarray analysisof gene expression in four breast cell lines [MCF-7 (ER+), T-47D (ER+),SK-BR-3 (ER−), and MDA-MB-231] following treatment with the syntheticLXR ligand GW3965. Id. In the common responsive gene set, upregulatedgenes tend to function in the known metabolic effects of LXR ligands andLXRs whereas the downregulated genes mostly include those which functionin cell cycle regulation, DNA replication, and other cellproliferation-related processes. Id. Transcription factor binding siteanalysis of the downregulated genes revealed an enrichment of E2Fbinding site sequence motifs. Id. Correspondingly, E2F2 transcriptlevels are downregulated following LXR ligand treatment. Id. Knockdownof E2F2 expression, similar to LXR ligand treatment, resulted in asignificant disruption of estrogen receptor positive breast cancer cellproliferation. Id. Ligand treatment also decreased E2F2 binding tocis-regulatory regions of target genes.

Expression of activated LXRα blocks proliferation of human colorectalcancer cells and slows the growth of xenograft tumors in mice, andreduces intestinal tumor formation after administration of chemicalcarcinogens in Apc(min/+) mice. Lo Sasso, G. et al., “Liver X receptorsinhibit proliferation of human colorectal cancer cells and growth ofintestinal tumors in mice,” Gastroenterology 144(7): 1497-507 (2013). Alink of LXRs to apoptosis has been reported. (Pehkonen, P. et al,“Genome-wide landscape of liver X receptor chromatin binding and generegulation in human macrophages,” BMC Genomics 13: 50 (2012)).

MicroRNAs and Alternative Splicing

Overall, relatively little has been reported on miRNAs in the context ofcholesterol synthesis. (Sharpe, L J and Brown, A J, “Controllingcholesterol synthesis beyond 3-hydroxy-3-methylglutaryl CoA reductase(HMGCR),” J. Biol. Chem. 288 (26): 18707-715 (2013)) In the context ofcholesterol metabolism, perhaps the best studied microRNA (miRNA) ismiR-33, an intronic miRNA encoded in the SREBP genes that controlscellular cholesterol export, whereas its SREBP host genes stimulatecholesterol synthesis (Id., citing Fernandez-Hernando, et al, “MicroRNAsin metabolic disease,” Arterioscl. Thromb. Vasc. Biol. 33: 178-85(2013)).

Alternative splicing of HMGCR is regulated by sterols, withproportionally less of an unproductive transcript present when sterollevels are low and more when sterol levels are higher (Id., citingMedina, M. W., et al, “Coordinately regulated alternative splicing ofgenes involved in cholesterol biosynthesis and uptake,” PLosONE 6:e19420 (2011)). This effect also extends to other cholesterogenic genes,including HMGSC1 and MVK (Id citing Medina, M. W., et al, “Coordinatelyregulated alternative splicing of genes involved in cholesterolbiosynthesis and uptake,” PLosONE 6: e19420 (2011)). Because the effectis mediated via SREBP-2 and alternative transcripts occur for allcholesterol synthesis enzymes beyond HMGCR (Id., citing de la Grange,P., et al, “a new advance in alternative splicing databases fromcatalogue to detailed analysis of regulation of expression and functionof human alternative splicing variants,” BMC Bioinformatics 8: 180(2007)), this effect may involve the entire cholesterol synthesispathway.

Post-Translational Regulation

Because transcriptional down-regulation via the SREBP pathway isrelatively slow, with mRNA of target genes decreasing only after severalhours, rapid shutdown of cholesterol synthesis requirespost-transcriptional control. Turnover of 3-hydroxy-3-methylglutaryl-CoAreductase (HMGCR) is accelerated by non-sterol and sterol products ofthe mevalonate pathways (Id., citing Roitelman, J. and Simoni, RD,“Distinct sterol and nonsterol signals for the regulated degradation of3-hyudroxy-3-methylglutaryl-CoA reductase,” J. Biol. Chem. 267:25264-273 (1992)), with physiological sterol degradation signals, suchas 24,25-dihydrolanosterol, and side chain oxysterols, such as 24,25-ECand 27-hydroxycholeseterol (generated from cholesterol itself (Id.,citing Lange, Y. et al, “Effectors of rapid homeostatic responses ofendoplasmic reticulum cholesterol and 3-hydroxy-3-methylglutaryl-CoAreductase,” J. Biol. Chem. 283: 1445-55 (2008); Nguyen, A D et al,“Hypoxyia stimulates degradation of 3-hydroxy-3-methylglutaryl-coenzymeA reductase through accumulation of lanosterol and hypoxia-induciblefactor-mediated induction of Insigs,” J. Biol. Chem. 282: 27436-446(2007)). The regulated turnover is proteosomal, and requires the Insigproteins, which also act to suppress SREBP activation (Jo, Y andDebose-Boyd, R A, “Control of cholesterol synthesis through regulatedER-associated degradation of HMG CoA reductase,” Crit. Rev. Biochem.Mol. Bio. 445: 185-198 (2010); Burg, J S and Espenshade, P J,“Regulation of HMG-CoA reductase in mammals and yeast,” Prog. Lipid Res.50: 403-410 (2011)).

Regulated ER-associated degradation also occurs for a later step incholesterol synthesis, catalyzed by squalene monooxygenase (SM), albeitby a mechanism distinct from HMGCR. Squalene monooxygenase has beenproposed as a second rate-limiting enzyme in cholesterol synthesis (Id.,citing Gonzalez, R. et al, “Two major regulatory steps in cholesterolsynthesis by human renal cancer cells,” Arch. Biochem. Biophys. 196:574-80 (1979); Hidaka, Y, et al, “Regulation of squalene epoxidase inHepG2 cells,” J. Lipid Res. 31: 2087-94 (1990)). Cholesterol itselfaccelerates SM degradation, an example of end product inhibition (Id.,citing Gill, S. et al, “Cholesterol-dependent degradation of squalenemonooxygenase, a control point in cholesterol synthesis beyond HMG-CoAreductase,” Cell Metab. 13: 260-73 (2011)), and unlike HMGCR, SMturnover does not require the Insig proteins.

Feedback Regulation of Cholesterol Synthesis

Cholesterol accumulation lowers the activity of HMG CoA reductase andseveral other enzymes in the cholesterol biosynthetic pathway, therebylimiting the production of cholesterol.

HMG CoA reductase, the rate-limiting enzyme in cholesterol synthesis,and the target of statins, is subject to feedback control throughmultiple mechanisms that are mediated by sterol and nonsterolend-products of mevalonate metabolism such that essential nonsterolisoprenoids can be constantly supplied without risking the potentiallytoxic overproduction of cholesterol or one of its sterol precursors.(DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis:Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,”Cell Res. 18 (6): 609-21 (2008)). For example, treatment of culturedcells with the statin Compactin, a competitive inhibitor of HMG-CoAreductase, blocks production of mevalonate, thereby reducing levels ofsterol and nonsterol isoprenoids that normally govern this feedbackregulation. Id. Cells respond to the inhibition of HMG-CoA reductasewith a compensatory increase in the reductase due to the combinedeffects of enhanced transcription of the reductase gene, efficienttranslation of mRNA, and extended half-life of reductase protein. Id.Complete reversal of this compensatory increase in reductase requiresregulatory actions of both sterol and nonsterol end-products ofmevalonate metabolism. Id.

Sterols inhibit the activity of sterol regulatory element-bindingproteins (SREBPs) and the low density lipoprotein (LDL)-receptor (Id.,citing Horton, J D, et al, “SREBPs: activators of the complete programof cholesterol and fatty acid synthesis in the liver,” J. Clin. Invest.109: 1125-31 (2002)). A nonsterol mevalonate-derived product(s)control(s) the translational effects through a poorly understoodmechanism that may be mediated by the complex 5′-untranslated region ofthe reductase mRNA (Id., citing Nakanishi, M. et al, “Multivalentcontrol of 3-hydroxy-3-methylglutaryl coenzyme A reductase.Mevalonate-derived product inhibits translation of mRNA and acceleratesdegradation of enzyme,” J. Biol. Chem. 263: 8929-37 (1988)). Both steroland nonsterol end-products of mevalonate metabolism combine toaccelerate degradation of reductase protein through a mechanism mediatedby the ubiquitin-proteosome pathway (Id., citing Roitelman, J. andSimoni, RD, “Distinct sterol and nonsterol signals for the regulateddegradation of 3-hydroxy-3-methylglutaryl-CoA reductase,” J. Biol. Che.267: 25264-273 (1992); McGee, T P et al, “Degradation of3-hydroxy-3-methylglutaryl-CoA reductase in endoplasmic reticulummembranes is accelerated as a result of increased susceptibility toproteolysis,” J. Biol. Chem. 271: 25630-638 (1996); Ravid, T. et al,“The ubiquitin proteasome pathway mediates the regulated degradation ofmammalian 3-hydroxy-3-methylglutaryl-Coenzyme A reductase,” J. Biol.Chem. 275: 35840-47 (2000)).

Inhibition of ER to Golgi transport of SREBPs results fromsterol-induced binding of SCAP to ER retention proteins calledinsulin-induced gene 1 and 2 proteins (Insig-1 and Insig-2)(DeBose-Boyd, R. A. “Feedback Regulation of Cholesterol Synthesis:Sterol-accelerated ubiquitination and degradation of HMG CoA Reductase,”Cell Res. 18 (6): 609-21 (2008., citing Yang, T. et al, “Crucial step incholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, amembrane protein that facilitates retention of SREBPs in ER,” Cell 110:489-500 (2002); Yabe, D. et al, “Insig-2, a second endoplasmic reticulumprotein that binds SCAP and blocks export of sterol regulatoryelement-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758(2002)). Insig binding occludes a cytosolic binding site in SCAPrecognized by COPII proteins, which incorporate cargo molecules intovesicles that deliver ER-derived proteins to the Golgi (Id., citing SunL P et al, “From the Cover: Sterol-regulated transport of SREBPs fromendoplasmic reticulum to Golgi: Insig renders sorting signal in Scapinaccessible to COPII proteins,” Proc. Natl Acad. Sci. USA 104: 6519-26(2007)). SCAP-Insig binding is mediated by a segment of SCAP's membranedomain that includes transmembrane helices 2-6 (Id., citing Hua, X etal, “Sterol resistance in CHO cells traced to point mutation in SREBPcleavage-activating protein,” Cell 87: 415-26 (1996); Yang, T. et al,“Crucial step in cholesterol homeostasis: sterols promote binding ofSCAP to INSIG-1, a membrane protein that facilitates retention of SREBPsin ER,” Cell 110: 489-500 (2002)), i.e., the sterol-sensing domain (Id.,citing Kuwabara, PE, “The sterol-sensing domain: multiple families, aunique role,” Trends Genet. 18: 193-201 (2002)), since a similar stretchof transmembrane helices is found in at least four other polytopicproteins, including the Niemann Pick C1 protein (part of an intestinalcholesterol transporter complex), Patched, Dispatched and reductase)that have been postulated to interact with sterols. Point mutationswithin this region disrupt Insig binding, which relieves sterol-mediatedretention of mutant SCAP-SREBP complexes in the ER (Id., citing Yang, T.et al, “Crucial step in cholesterol homeostasis: sterols promote bindingof SCAP to INSIG-1, a membrane protein that facilitates retention ofSREBPs in ER,” Cell 110: 489-500 (2002); Yabe, D., “Insig-2, a secondendoplasmic reticulum protein that binds SCAP and blocks export ofsterol regulatory element-binding proteins,” Proc. Natl. Acad. Sci. USA99: 12753-758 (2002); Yabe, D. et al, “Three mutations in sterol-sensingdomain of SCAP block interaction with insig and render SREBP cleavageinsensitive to sterols,” Proc. Natl Acad. Sci. USA 99: 16672-77 (2002);Nohturfft, A. et al, “A substitution in a single codon of SREBPcleavage-activating protein causes sterol resistance in three mutantChinese hamster ovary cell lines,” Proc. Natl Acad. Sci. USA 93:13709-714 (1996); Nohturfft, A. et al, “Sterols regulate processing ofcarbohydrate chains of wild-type SREBP cleavage-activating protein(SCAP), but not sterol-resistant mutants Y298C o D443N,” Proc. NatlAcad. Sci. USA 95: 12848-853 (1998)).

The following observations suggest that Insigs may play a role indegradation of HMG CoA reductase. First, when Insigs are overexpressedby transfection in Chinese hamster ovary (CHO) cells, HMG CoA reductasecannot be degraded when the cells are treated with sterols (Id., citingSever, N. et al, “Accelerated degradation of HMG CoA reductase mediatedby binding of insig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33(2003)). Co-expression of Insig-1 restores sterol-accelerateddegradation of HMG CoA reductase, suggesting the saturation ofendogenous Insigs by the overexpressed reductase. Id. Second, reductionof both Insig-1 and Insig-2 by RNA interference (RNAi) abolishessterol-accelerated degradation of endogenous HMG CoA reductase (Id.,citing Sever, N. et al, “Insig-dependent ubiquitination and degradationof mammalian 3-hydroxy-3-methylglutaryl CoA reductase stimulated bysterols and geranylgeraniol,” J. Biol. Chem. 278: 52479-90 (2003)).Third, mutant CHO cells lacking both Insigs are impervious tosterol-stimulated degradation of HMG CoA reductase as well assterol-mediated inhibition of SREBP processing (Id., citing Lee, P. C.et al, “Isolation of sterol-resistant Chinese hamster ovary cells withgenetic deficiencies in both Insig-1 and Insig-2,” J. Biol. Chem. 280:25242-249 (2005)).

Degradation of HMG CoA reductase coincides with sterol-induced bindingof its membrane domain to Insigs (Id., citing Sever, N. et al,“Accelerated degradation of HMG CoA reductase mediated by binding ofinsig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003)), anaction that requires a tetrapeptide sequence (YIYF) located in thesecond transmembrane segment of HMG CoA reductase. A mutant form of HMGCoA reductase in which the YIYF sequence is mutated to alanine residuesno longer binds to Insigs, and the enzyme is not subject to rapiddegradation. The YIYF sequence is also present in the secondtransmembrane domain of SCAP, where it mediates sterol-dependentformation of SCAP-Insig complexes (Id., citing Yang, T. et al, “Crucialstep in cholesterol homeostasis: sterols promote binding of SCAP toINSIG-1, a membrane protein that facilitates retention of SREBPs in ER,”Cell 110: 489-500 (2002); Yabe, D., “Insig-2, a second endoplasmicreticulum protein that binds SCAP and blocks export of sterol regulatoryelement-binding proteins,” Proc. Natl. Acad. Sci. USA 99: 12753-758(2002)). Overexpressing the sterol-sensing domain of SCAP in cellsblocks Insig-mediated, sterol-accelerated degradation of HMG CoAreductase; mutation of the YIYF sequence in the SCAP sterol-sensingdomain ablates this inhibition, suggesting that SCAP and HMG CoAreductase bind to the same site on Insigs and that the two proteinscompete for limiting amounts of Insigs when intracellular sterol levelsrise. Id.

Glycoprotein 78 (Gp78), an E3 ubiquitin ligase, mediates ubiquitinationof ApoB-100, Insig 1 and 2 proteins, and HMG-CoA reductase (Jiang, W.,Song, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,” DiabetesMetab. 38: 171-80 (2014)). High concentration of sterol (lanosterol)promote the NH₂-terminal transmembrane domain of3-hydroxy-3-methylglutaryl CoA reductase to interact with Insigs (Id.,citing Sever, N. et al, “Accelerated degradation of HMG CoA reductasemediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell11: 25-33 (2003); Song, B L, et al, “Insig-mediated degradation of HMGCoA reductase stimulated by lanosterol, an intermediate in the synthesisof choleseterol,” Cell Metab. 1: 179-89 (2005)), and sterol-dependentInsig binding results in recruitment of ubiquitin ligase.

Gp78 binds Insig-1 constitutively in the ER membrane. Id. When thecellular sterol level is high, the insig-1/gp78 complex binds thetransmembrane domain of 3-hydroxy-3-methylglutaryl CoA reductase. Id.With the assistance of at least two proteins associated with gp78,p97/VCP and Aup1 (Id., citing Song, B L et al, “Gp8, a membrane-anchoredubiquitin ligase, associates with Insig-1 and couples sterol-regulatedubiquitination to degradation of HMG CoA reductase,” Mol. Cell 19:829-40 (2005); Jo, Y et al, “ancient ubiquitous protein 1 mediatessterol-induced ubiquitination of 3-hydroxy-3-methylglutaryl CoAreductase in lipid droplet-associated endoplasmic reticulum membranes,”Mol. Biol. Cell 24: 169-83 (2013)), the ubiquitinated reductase istranslocated to lipid droplet-associated ER membrane and dislocated frommembrane into cytosol for proteosomal degradation (Id., citing Jo, Y etal, “ancient ubiquitous protein 1 mediates sterol-induced ubiquitinationof 3-hydroxy-3-methylglutaryl CoA reductase in lipid droplet-associatedendoplasmic reticulum membranes,” Mol. Biol. Cell 24: 169-83 (2013);Hartman I Z, et al, “Sterol-induced dislocation of3-hydroxy-3-methylglutaryl coenzyme A reductase from endoplasmicreticulum membranes into the cytosol through a subcellular compartmentresembling lipi droplets,” J. Biol. Chem. 285: 19288-98 (2010)). Thispost-ubiquitination process can be promoted by geranylgeraniol or itsmetabolically active geranyl-geranyl-pyrophosphate (Id., citing Sever,N. et al, “Insig-dependent ubiquitination and degradation of mammalian3-hydroxy-3-methylglutaryl CoA reductase stimulated by sterols andgeranylgeraniol,” J. Biol. Chem. 278: 52479-90 (2003)).

In short, the ubiquitination of Insig-1 is mediated by gp78 andregulated by sterols. Id. Insig-1 is modified by gp78 under low sterolconditions. Id. High sterol promotes SCAP to bind Insig and gp78 iscompeted off, thereby stabilizing Insig-1. Id.

Gp78-mediated ubiquitination and degradation of Insig-1 provides amechanism for convergent feedback inhibition, whereby inhibition ofSREBP processing requires convergence of newly synthesized Insig-1 andnewly acquired sterols. (DeBose-Boyd, R. A. “Feedback Regulation ofCholesterol Synthesis: Sterol-accelerated ubiquitination and degradationof HMG CoA Reductase,” Cell Res. 18 (6): 609-21 (2008); citing Gong, Y.et al, “Sterol-regulated ubiquitination and degradation of Insig-1creates a convergent mechanism for feedback control of cholesterolsynthesis and uptake,” Cell Metab. 3: 15-24 (2006)). In sterol-depletedcells, SCAP-SREBP complexes no longer bind Insig-1, which in turnbecomes ubiquitinated and degraded. Id. These SCAP-SREBP complexes arefree to exit the ER and translocate to the Golgi, where the SREBPs areprocessed to the nuclear form that stimulates transcription of targetgenes, including the Insig-1 gene. Id. Increased transcription of theInsig-1 gene leads to increased synthesis of Insig-1 protein, but theprotein is ubiquitinated and degraded until sterols build up to levelssufficient to trigger SCAP binding. Id.

Insig-2 has been defined as a membrane-bound oxysterol binding proteinwith binding specificity that correlates with the ability of oxysterolsto inhibit SREBP processing (Id., citing Sun, L P, et al,“Sterol-regulated transport of SREBPs from endoplasmic reticulum toGolgi: Insig renders sorting signal in Scap inaccessible to COPIIproteins,” Proc. Natl Acad. Sci. USA 104: 6519-26 (2007); Radhakrishnan,A. et al, “From the Cover: Sterol-regulated transport of SREBPs fromendoplasmic reticulum to Golgi: Oxysterols block transport by binding toInsig,” Proc. Natl Acad. Sci. USA 104: 6511-18 (2007)). Oxysterols,cholesterol derivatives that contain hydroxyl groups at variouspositions in the iso-octyl side chain (e.g., 24-hydroxycholesterol,25-hydroxycholesterol, 27-hydroxycholesterol), are synthesized in manytissues by specific hydrolases; oxysterols play key roles in cholesterolexport, and are intermediates in the synthesis of bile acids (Id.,citing Russell, D W, “Oxsterol biosynthetic enzymes,” Biochim. Biophys.Acta—Molec. Cell Biol. Lipids 1529: 126-135 (2000)). Oxysterols, whichare significantly more soluble than cholesterol in aqueous solution, canreadily pass across the plasma membrane and enter cells, and areextremely potent in inhibiting cholesterol synthesis by stimulatingbinding of both HMG Co A reductase and SCAP to Insigs. Id. Thus,formation of the SCAP-Insig complex can be initiated by either bindingof cholesterol to the membrane domain of SCAP or by binding ofoxysterols to Insigs, both of which prevent incorporation of SCAP-SREBPinto vesicles that bud from the ER en route to the Golgi. Id.

Insig-mediated regulation of HMG Co A reductase is controlled by threeclasses of sterols: oxysterols, cholesterol, and methylated sterols(e.g., lanosterol and 24, 25-dihydrolanosterol). Id. Oxysterols bothaccelerate degradation of HMG Co A reductase and block ER to Golgitransport of SCAP-SREBP through their direct binding to Insigs. Id.Cholesterol does not regulate HMG Co A reductase stability directly, butbinds to SCAP and triggers Insig binding, thereby preventing escape ofSCAP-SREBP from the ER.Id. Lanosterol selectively acceleratesdegradation of HMG Co A reductase without an effect on ER to Golgitransport of SCAP-SREBP. Id. However, the demethylation of lanosterolhas been implicated as a rate-limiting step in the post-squalene portionof cholesterol synthesis (Id., citing Gaylor, J L, “Membrane boundenzymes of cholesterol synthesis from lanosterol,” Biochem. Biophys.Res. Communic., 292: 1139-46 (2002); Williams, M T, et al,“Investigation of the rate-determining microsomal reaction ofcholesterol biosynthesis from lanosterol in Morris hepatomas and liver,”Cancer Res. 37: 1377-83 (1977)). The accumulation of lanosterol isavoided; its inability to block SREBP processing through SCAP assuresthat mRNAs encoding enzymes catalyzing reactions subsequent tolanosterol remain elevated, and lanosterol is metabolized tocholesterol.

It is a paradox that gp78 deficiency increases both the3-hydroxy-3-methylglutaryl CoA reductase and Insig protein levels inmouse liver, because Insigs not only negatively regulate3-hydroxy-3-methylglutaryl CoA reductase post-transcriptionally, butalso inhibit SREBPs processing through binding to SCAP (Jiang, W. andSong, B-L, “Ubiquitin Ligases in Cholesterol Metabolism,” DiabetesMetab. 38: 171-80 (2014) citing Nohturfft, A. et al., “Topology of SREBPcleavage-activating protein, a polytopic membrane protein with a sterolsensing domain,” J. Biol. Chem. 273: 17243-250 (1998)). These twooutcomes are contradictory regarding cholesterol biosynthesis. Studiesfrom L-gp78+ mice have shown that the biosynthesis of cholesterol andfatty acids is decreased in gp78-deficient mouse liver (Id., citingEdwards, P A et al, “Purification and properties of rat liver3-hydroxy-3-methylglutaryl coenzyme A reductase,” Biochim. Biophys. Acta574: 123-35 (1979)). This has been interpreted to mean that theInsig-SCAP-SREBP axis dominates, even though 3-hydroxy-3-methylglutarylCoA (HMG CoA) reductase is elevated. Id.

ApoB-100, an essential protein component of very low densitylipoproteins (VLDL) and low density lipoproteins (LDL), which playscritical roles in plasma cholesterol transportation, is anothersubstrate of g78. Id. Under normal conditions, ApoB-100 is one of thecommitted secretory proteins. Id. However, when the cellular lipidavailability is limited (e.g., the new synthesized core lipids(triglyceride, cholesterol ester) or microsomal triglyceride transferprotein activity is decreased), the nascent ApoB-100 is subjected toER-associated degradation mediated by gp78. Id. When gp78 isoverexpressed, ubiquitination and degradation through the 26S proteosomeof apoB-100 is decreased (Id., citing Ravid, T. et al, “Theubiquitin-proteasome pathway mediates the regulated degradation ofmammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase,” J. Biol.Chem. 275: 35840-847 (2000)). When gp78 is knocked down, the secretionof apoB-100 and the assembly of VLDL are increased in HepG2 cells (Id.,citing Hua, X., et al, “Sterol resistance in CHO cells traced to pointmutation in SREBP cleavage-activating protein,” Cell 87: 415-426(1996)). The retrotranslocation of ApoB-100 also requires p97/VCP,similar to HMG CoA reductase (Id, citing Nakanishi, M. et al,“multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase.Mevalonate-derived product inhibits translation of mRNA and acceleratesdegradation of enzyme,” J. Biol. Chem. 263: 8929-37 (1988); Hua, X., etal, “Sterol resistance in CHO cells traced to point mutation in SREBPcleavage-activating protein,” Cell 87: 415-426 (1996)).

TRC8

Human TRC8 is a multi-pass membrane protein located in the ER membranethat binds both Insig-1 and Insig-2. (Id., citing Inoue, S. et al,“Inhibition of degradation of 3-hydroxyl-3-methylglutaryl-coenzyme Areductase in vivo by cysteine protease inhibitors,” J. Biol. Chem. 266:13311-17 (1991)). It contains a conserved sterol sensing domain andC-terminal RING domain with ubiquitin ligase activity (Id., citing Yabe,D. et al, “Insig-2, a second endoplasmic reticulum protein that bindsSCAP and blocks export of sterol regulatory element-binding proteins,”Proc. Natl. Acad. Sci. USA 99: 12753-758 (2002); Sun L P et al, “Fromthe Cover: Sterol-regulated transport of SREBPs from endoplasmicreticulum to Golgi: Insig renders sorting signal in Scap inaccessible toCOPII proteins,” Proc. Natl Acad. Sci. USA 104: 6519-26 (2007)). RNAistudies in SV-589 cells showed that knockdown of TRC8 combined with gp78can dramatically decrease the sterol-regulated ubiquitination as well asdegradation of HMG CoA reductase, suggesting that both gp78 and TRC8 areinvolved in the sterol-accelerated ubiquitination of HMG CoA reductasein CHO-7 and SV-589 cells. (Id., citing Inoue, S. et al, “Inhibition ofdegradation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase in vivoby cysteine protease inhibitors,” J. Biol. Chem. 266: 13311-17 (1991)).

TEB4

Human TEB4 is a 910 amino acid ER membrane-resident ubiquitin ligase. Inmammalian cells, cholesterol stimulates the degradation of squalenemonooxygenase (SM), the enzyme that catalyzes the first oxygenation stepin cholesterol synthesis by which squalene is converted to thesqualene-2,3-epoxide (37) mediated by TEB4 (Id., citing Sever, N. et al,“Accelerated degradation of HMG CoA reductase mediated by binding ofinsig-1 to its sterol-sensing domain,” Mol. Cell 11: 25-33 (2003)). Asone of the target genes of SREBP-2, both the transcription of SM and thestability of SM protein are regulated by sterols (Id., citing Sever, N.et al, Insig-dependent ubiquitination and degradation of mammalin3-hydroxy-3-methylglutaryl-CoA reductase stimulated by sterols andgeranylgeraniol,” J. Biol. Chem. 278: 52479-490 (2003)). SM proteinlevel is negatively regulated by cholesterol in mammalian cells (Id.,citing Lee, P C, et al, “Isolation of sterol-resistant Chinese hamsterovary cells with genetic deficiencies in both Insig-1 and Insig-2,” J.Biol. Chem. 280: 25242-249 (2005)). When cholesterol, but not 24,25-dihydrolanosterol, or side chain oxysterols, such as27-hydroxycholesterol, is/are present, SM is ubiquitinated by TEB4 (Id.,citing Sever, N. et al, “Accelerated degradation of HMG CoA reductasemediated by binding of insig-1 to its sterol-sensing domain,” Mol. Cell11: 25-33 (2003); Lee, P C, et al, “Isolation of sterol-resistantChinese hamster ovary cells with genetic deficiencies in both Insig-1and Insig-2,” J. Biol. Chem. 280: 25242-249 (2005)).

IDOL

The low density lipoprotein receptor (LDL-R) gene family consists ofcell surface proteins involved in receptor-mediated endocytosis ofspecific ligands. Low density lipoprotein (LDL) is normally bound at thecell membrane and taken into the cell, ending up in lysosomes where theprotein is degraded and the cholesterol is made available for repressionof microsomal enzyme HMG CoA reductase. At the same time, a reciprocalstimulation of cholesterol ester synthesis takes place.

Inducible degrader of LDL-R (IDOL) moderates the degradation of LDL-Rand requires the E2 enzyme UBE2D (Id., citing Schroepfer, G J, Jr.,“Oxysterols: modulators of cholesterol metabolism and other processes,”Physiol. Rev. 80: 361-554 (2000); Bjorkhem, I., “Do oxysterols controlcholeseterol homeostasis,” J. Clin. Invest. 110: 725-30 (2002)).

Transcription of the LDL-R gene is regulated primarily by SREBP in asterol responsive manner. (Id.) The LDL-R is also regulated at theposttranscriptional level by protoprotein convertase subtilisin/kexintype 9 (PCSK9)-mediated degradation of LDLR in the lysosome. (Id.,citing Radhakrishnan, A. et al, “Direct binding of cholesterol to thepurified membrane region of SCAP: Mechanism for a sterol-sensingdomain,” Mol. Cell 15: 259-68 (2004)). PCSK9 is synthesized as an about74 kD soluble zymogen in the endoplasmic reticulum (ER), where itundergoes autocatalytic processing to release a processing enzyme ofabout 60 kDa to secrete from cells. (Id.) PCSK9 binds the extracellulardomain of LDLR, which leads to lysosomal degradation of LDLR. (Id.)

IDOL also is a post-transcriptional regulator of LDL-R (Id., citingSchroepfer, G J, Jr., “Oxysterols: modulators of cholesterol metabolismand other processes,” Physiol. Rev. 80: 361-554 (2000)). Activation ofLXR can decrease the abundance of LDLR without changing its mRNA leveland subsequently inhibited uptake of LDL in different cells (Id., citingSchroepfer, G J, Jr., “Oxysterols: modulators of cholesterol metabolismand other processes,” Physiol. Rev. 80: 361-554 (2000)). IDOL canincrease plasma cholesterol level by ubiquitination and degradation ofLDL-R dependent on its cytosolic domain. The decrease or ablation ofIDOL can elevate the LDL-R protein level and promote LDL uptake. Theexpression of Idol in liver is relatively low, and it is not regulatedby LXR, while the LXR-IDOL pathway seems to be more active in peripheralcells, e.g., macrophages, small intestine, adrenals.

Cholesterol Biosynthesis Pathway Inhibitors as Antitumor Agents

Statins, which were developed as lipid-lowering drugs to controlhypercholesterolemia, competitively inhibit HMG-CoA reductase, and havebeen proposed as anticancer agents, because of their ability to triggerapoptosis in a variety of tumor cells in a manner that is sensitive andspecific to the inhibition of HMG-CoA reductase (Thumher, M., et al.,“Novel aspects of mevalonate pathway inhibitors as antitumor agents,”Clin. Cancer Res. 18: 3524-31 (2012) citing Wong, W W et al, “HMG-CoAreductase inhibitors and the malignant cell: the statin family of drugsas triggers of tumor-specific apoptosis,” Leukemia 16: 508-19 (2002)).This apoptotic response is in part due to the downstream depletion ofgeranylgeranyl pyrophosphate (GGPP), and thus due to inhibition ofprotein prenylation. Protein prenylation creates a lipidated hydrophobicdomain and plays a role in membrane attachment or protein-proteininteractions. Prenylation occurs on many members of the Ras and Rhofamily of small guanosine triphosphatases (GTPases). Three enzymes(farnesyltransferase (FTase), geranylgeranyltransferase (GGTase) I andGGTase II can catalyze protein prenylation.

While statin therapy blocks the intracellular synthesis of cholesterol,it also alters the cholesterol content of tumor cell membranes,interfering with key signaling pathways. (Cruz, P M R, et al, “The roleof cholesterol metabolism and cholesterol transport in carcinogenesis: areview of scientific findings, relevant to future cancer therapeutics,”Frontiers in Pharmacol. 4(119): doi:10.3369/phar.2013.00119, citingZhuang, L. et al, “Cholesterol targeting alters lipid raft compositionand cell survival in prostate cancer cells and zenografts,” J. Clin.Invest. 115: 959-68 (2005)).

Statins have been shown to have immunomodulatory activity (Thumher, M.,et al., “Novel aspects of mevalonate pathway inhibitors as antitumoragents,” Clin. Cancer Res. 18: 3524-31 (2012), citing Greenwood, J etal, Statin therapy and autoimmune disease: from protein prenylation toimmunomodulation,” Nat. Rev. Immunol. 6: 358-70 (2006)), and to inducethe depletion of prenyl pyrophosphates in human dendritic cells[Gruenbacher, G. et al., “CD56+ human blood dendritic cells effectivelypromote TH1-type gammadelta T cell responses,” Blood 114: 4422-31(2009); Steinman, R M, Banchereau, J., “Taking dendritic cells intomedicine,” Nature 449: 419-26 (2007)). Prenyl pyrophosphate deprivationtranslated into activation of caspase I, which cleaved the preforms ofIL-1β and IL-18 and enabled the release of bioactive cytokines. Thestatin-treated dendritic cells (DCs) thus acquired the capability topotentially activate IL-2 primed natural killer (NK) cells (Id., citingGruenbacher, G. et al., “IL-2 costimulation enables statin-mediatedactivation of human NK cells, preferentially through a mechanisminvolving CD56+ dendritic cells,” Cancer Res. 70: 9611-20 (2010)). NKcells, which recognize and attack tumor cells that lack MHC class Imolecules (Id., citing Munz, C. et al, “Dendritic cell maturation byinnate lymphocytes: coordinated stimulation of innate and adaptiveimmunity,” J. Exptl Med. 202: 203-7 (2005); Maniar, A. et al, “Humangammadelta T lymphocytes induce robust NK cell-mediated antitumorcytotoxicity through CD137 engagement,” Blood 116: 1726-33 (2010))contribute to innate immune responses against neoplastic cells. Thestatin-induced response of IL-2-primed NK cells could be abolishedcompletely when cell cultures were reconstituted with the isoprenoidpyrophosphate GGPP, which allows protein geranylgeranylation to occurdespite statin-mediated inhibition of HMB-CoA reductase. Statins alsoacted directly on human carcinoma cells to induce apoptosis, and IFN-γproduced by NK cells cooperated with statins to enhance tumor cell deathsynergistically (Id., citing Gruenbacher, G. et al., “IL-2 costimulationenables statin-mediated activation of human NK cells, preferentiallythrough a mechanism involving CD56+ dendritic cells,” Cancer Res. 70:9611-20 (2010)).

Mutant p53, which is present in more than half of all human cancers, cansignificantly upregulate mevalonate pathway activity in cancer cells,which contributes to maintenance of the malignant phenotype. (Id.,citing Freed-Pastor, W A, et al, “Mutant p53 disrupts mammary tissuearchitecture via the mevalonate pathway,” Cell 148: 244-58 (2012)).Simvastatin was shown to reduce 3-dimensional growth of cancer cellsexpressing a single mutant p53 allele, and was able to induce extensivecancer cell death and a significant reduction of their invasivephenotype. In isoprenoid add-back experiments, supplementation with GGPPwas sufficient to restore the invasive phenotype in the presence ofHMG-CoA reductase inhibition, showing that upregulation of proteingeranylgeranylation is an important effect of mutant p53 (Id., citingFreed-Pastor, W A, et al, “Mutant p53 disrupts mammary tissuearchitecture via the mevalonate pathway,” Cell 148: 244-58 (2012)).

Bisphosphonates, drugs that prevent bone resorption, act downstream ofHMG-CoA reductase to inhibit farnesyl pyrophosphate (FPP) synthase. Bothbisphosphonates and statins eventually cause FPP and GGPP deprivationand thus failure to perform farnesylation and geranylgeranylation ofsmall GTPases of the Ras superfamily. With regard to bisphosphonates,the inhibition of Ras signaling due to the disruption of membraneanchoring of these GTPases eventually stops osteoclast-mediated boneresorption (Id., citing Konstantinopoulos, P A, et al, “Posttranslational modifications and regulation of the RAS superfamily ofGTPases as anticancer targets,” Nat. Rev. Drug Discov. 6: 541-55(2007)).

Suppressors of the mevalonate pathway also include the diverseisoprenoids (Cruz, P M R, et al, “The role of cholesterol metabolism andcholesterol transport in carcinogenesis: a review of scientificfindings, relevant to future cancer therapeutics,” Frontiers inPharmacol. 4(119): doi:10.3369/phar.2013.00119, Id., citing Mo, H andElson, C E, “Studies of the isopreoid-mediated inhibition of mevalonatesynthesis applied to cancer chemotherapy and chemoprevention,” Exp.Biol. Med. (Maywood) 229: 567-85 (2004)), mevalonate-derived secondarymetabolites of plants (Bach, T J, “Some new aspects of isoprenoidbiosynthesis in plants—a review,” Lipids 30: 191-202 (1995)). Thepotencies of isoprenoids in suppressing hepatic HMG-CoA reductaseactivity was found to be strongly correlated to their potencies in tumorsuppression (Id., citing Elson, C E and Quereshi, A A, “Coupling thecholeseterol—and tumor-suppressive actions of palm oil to the impact ofits minor constituents on 3-hydroxy-3-methylglutaryl coenzyme Areductase activity,” Prostaglandins Leukot. Essent. Fatty Acids 52:205-207 (1995)). The tocotrienols, vitamin E molecules, and “mixedisoprenoids” with a farnesol side chain, down-regulate HMG-CoA reductaseactivity in tumors and consequently induce cell cycle arrest andapoptosis (Id., citing Mo H and Elfakhani, C E, “Mevalonate-suppressivetocotrienols for cancer chemoprevention and adjuvant therapy, inTocotrienols: Vitamin E beyond tocopherols, eds. RR. Wilson et al (BocaRaton: CRC Press), 135-149 (2013)). The growth-suppressive effect oftocotrienols was attenuated by supplemental mevalonate (Id., citingHussein, D and Mo, H, “d-δ-tocotrienol-mediated suppression of theproliferation of human PANC-1, M1A PaCa2 and BxPC-3 pancreatic carcinomacells,” Pancreas 38: e124-e136 (2009)).

Activity of azole antifungal compounds, such as ketoconazole, to blockthe function of several cytochrome P450 enzymes involved in cholesterolbiosynthesis (e.g., CYP51A1, which catalyzes demethylation oflanosterol) and CYP17A1 (which mediates a step in the synthesis ofandrogens) has been utilized clinically to treat hormone refractoryprostate cancer, and recently has been surpassed by abiraterone, aCYP17A1 antagonist. Gorin, A. et al., “Regulation of cholesterolbiosynthesis and cancer signaling,” Curr. Op. Pharmcol. 12(6) 710-16(2012); citing (4). Itraconazole has shown activity againstmedulloblastoma, via its inhibitory effects on Smoothened in thehedgehog pathway. (Id., citing Kim, J. et al, “Itraconazole, a commonlyused antifungal that inhibits Hedgehog pathway activity and cancergrowth,” Cancer Cell. 17(4): 388-99 (2010)), and suppression ofangiogenesis via its interference with lysosomal cholesterol trafficking(Id., citing Xu, J. et al, “Choleserol trafficking is required for mTPORactivation in endothelial cells,” Proc. Natl Acad. Sci. USA 107(10):4764-69 (2010)). The anti-angiogenic effect of itraconazole, awell-established CYP51/ERG11 antifungal antibiotic, is exerted viainhibition of endosomal cholesterol trafficking and suppression of mTORsignaling (Id.).

In tumor cells, increased signaling activity of growth factor or steroidhormone receptors via PI3K/AKT and MAPK/ERK1/2 (Gorin, A. et al.,Regulation of choletrol biosynthesis and cancer signaling, Curr. Opin.Pharmacol. 12(6): 710-16 (2012), citing Menendez, J A and Lupu, R.,Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis,”Nat. Rev. Cancer 7(10): 763-77 (2007)), HIF-1a, p53 (Id., citingOliverase, G. et al, “Novel anti-fatty acid synthase compounds withanti-cancer activity in Her2+ breast cancer,” Ann. N.Y. Acad. Sci. 1210:86-92 (2010)) and sonic hedgehog (SHH) (Id., citing Bhatia, B. et al,“Sonic hedgehog signaling and malignant transformation of the cerebellargranule neuron precursor cells,” Oncogene 30(4): 410-22 (2011)) pathwaysmodulate and activate SREBP-1, the main regulatory component oflipogenesis. It has been reported that inhibiting mTORC1 using rapamycinhas little effect on SREBP-1 nuclear localization and its abundance, butinhibiting its upstream factors, like EGFR, PI3K and Akt, significantlydecreases SREBP-1 N-terminal levels and diminishes its abundance in thenucleus (Guo, D et al, “Targeting SREBP-1 driven lipid metabolism totreat cancer,” Curr. Pharm Des. 20(15): 2619-26 (2014) citing Guo, D. etal, “EGFR signaling through han Akt-SREBP-1-dependent,rapamycin-resistant pathway sensitizes glioblastomas to antilipogenictherapy,” Science Signaling 2: ra82 (2009)). mTOR kinase inhibitorTorin-1 (Id., citing Peterson, T R et al, “DEPTOR is an mTOR inhibitorfrequently overexpressed in multiple myeloma cells and required fortheir survival,” Cell 137: 873-86 (2009)), which inhibits both mTORC1and mTORC2 activity (Id., citing Sabatini, D M, “mTOR and cancer:insights into a complex relationship,” Nat. Rev. Cancer 6: 729-34(2006)), significantly decreased SREBP-1 abundance in the nucleuscompared to the inhibition of mTORC1 alone by rapamycin (Id., citingPeterson, T R, et al, “mTOR complex I regulates lipin 1 localization tocontrol the SREBP pathway,” Cell 146: 408-20 (2011), Hagiwara, et al,“Hepatic mTORC2 activates glycolysis and lipogenesis through Akt,glucokinase and SREBP1c,” Cell Metab. 15: 725-38 (2012)).

Overexpression of lipogenic enzymes has been observed in a number ofcarcinomas (Gorin, A. et al., Regulation of cholesterol biosynthesis andcancer signaling, Curr. Opin. Pharmacol. 12(6): 710-16 (2012), citingNagahashi, M. et al, “Sphingosine-1-phosphate produced by sphingosinekinase 1 promotes breast cancer progression by stimulating angiogenesisand lymphangiogenesis,” Cancer Res. 72(3): 726-35 (2012)) and has beendescribed to correlate with disease severity, increased risk ofrecurrence and a lower chance of survival (Id., citing Uddin, S. et al,“High prevalence of fatty acid synthase expression in colorectal cancersin Middle Eastern patients and its potential role as a therapeutictarget,” Am. J. Gastroenterol. 104(7): 1790-1801 (2009; Mashima, T. etal, “De novo fatty-acid synthesis and related pathways as moleculartargets for cancer therapy,” Br. J. Cancer 100 (9): 1369-72 (2009)).

Accelerated synthesis of lipids and sterols also is an essentialmechanistic component of malignant transformation. Oxidized LDL receptor1 (OLR1) is required for Src kinase transformation of immortalizedMCF10A mammary epithelial cells (Id., citing Hirsch, H A et al, “Atranscriptional signature and common gene networks link cancer withlipid metabolism and diverse human diseases,” Cancer Cell. 17(4): 348-61(2010)). OLR1 is significantly induced during transformation, anddepletion of OLR1 by siRNA blocks morphological transformation andinhibits cell migration and invasion, and results in reduction of tumorgrowth in vivo (Id.). Conversely, overexpression of ORL1 protein inMCF10A and HCC1143 mammary epithelial cells leads to significantupregulating of BCL2, a negative regulator of apoptosis (Id., citingKhaidakov, M., et al., “Oxidized LDL receptor 1 (OLR1) as a possiblelink between obesity, dyslipidemia and cancer,” PLoS One 6(5): e20277(2011)).

EBP in complex with dihydrocholesterol-7 reductase (DHCR7) catalyzesisomerization of the double-bond between C7 and C8 in the secondcholesterol ring. (Gabitova, L. et al., “Molecular Pathways: Sterols andreceptor signaling in Cancer,” Clin. Cancer Res. 19(23): 6344-50(2013)). This complex mediates the activity of cholesterol epoxidehydrolase (Id., citing de Medina, P. et al, “Identification andpharmacological characterization of choleseterol-5,6-epoxide hydrolaseas a target for tamoxifen and AEBS ligands,” Proc. Natl. Acad. Sci. USA107: 13520-5 (2010)).

There are several known inhibitors of EBP, and some have been describedas anti-cancer agents. For example, a sterol conjugate of a naturallyoccurring steroidal alkaloid,5alpha-hydroxy-6beta-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3beta-ol(dendrogenin A) which is produced in normal, but not in cancer cells,and 5,6 alpha-epoxy-cholesterol and histamine (Id., citing de Medina, P.et al, “Dendrogenin A arises from cholesterol and histamine metabolismand shows cell differentiation and anti-tumour properties,” NatureCommunic. 4: 1840 (2013); de Medina, P. et al, “Synthesis of newalkylaminooxysterols with potent cell differentiating activities:identification of leads for the treatment of cancer andneurodegenerative diseases,” J. Med. Chem. 52: 7765-77 (2009)), has beenshown to suppress cancer cell growth and to induce differentiation invitro in various tumor cell lines of different types of cancers (Id.,citing de Medina, P. et al, “Synthesis of new alkylaminooxysterols withpotent cell differentiating activities: identification of leads for thetreatment of cancer and neurodegenerative diseases,” J. Med. Chem. 52:7765-77 (2009)). It also inhibited tumor growth in melanoma xenograftstudies in vivo and prolonged animal survival. (Id., citing de Medina,P. et al, “Dendrogenin A arises from cholesterol and histaminemetabolism and shows cell differentiation and anti-tumour properties,”Nature Communic. 4: 1840 (2013);).

SR31747A(cis-N-cyclohexyl-N-ethyl-3-(3-choloro-4-cyclohexyl-phenyl)propen-2-ylaminehydrochloride), a selective peripheral sigma binding site ligand whosebiological activities include immunoregulation and inhibition of cellproliferation, binds to SR31747A-binding protein 1 (SR-BP) and EBP withnanomolar affinity. Berthois, Y. et all., “SR31747A is a sigma receptorligand exhibiting antitumoural activity both in vitro and in vivo,” Br.J. Cancer 88: 438-46 (2003). The effect of SR31747A on proliferativeactivity was evaluated in vitro on the following breast and prostatecancer cell lines: breast (hormone responsive MCF-7 cells from a breastadenocarcinoma pleural effusion; MCF-7AZ; Hormone independent MCF-7/LCC1cells derived from MCF-7 cell lines; MCF-7LY2, resistant to thegrowth-inhibitory effects of the antiestrogen LY117018; Hormoneunresponsive MDA-MB-321 and BT20 established from a metastatic humanbreast cancer tumor); and prostate (Hormone responsive prostate cancercell line LNCaP; hormone-unresponsive PC3 cell line established frombone marrow metastasis; hormone-unresponsive DU145 established frombrain metastasis). Id. SR31747A induced concentration-dependentinhibition of cell proliferation, regardless of whether the cells werehormone responsive or unresponsive. Id. The antiproliferative effect ofSR31747A was partially reduced by adding cholesterol (Id.; Labit-LeBouteiller, C. et al., “Antiproliferative effects of SR31747A in animalcell lines are mediated by inhibition of cholesterol biosynthesis at thesterol isomerase step,” Eur. J. Biochem. 256: 342-49 (1998)), thusdemonstrating the involvement of EBP. Sensitivity to SR31747A did notcorrelate with cellular levels of EBP. Berthois, Y. et all., “SR31747Ais a sigma receptor ligand exhibiting antitumoural activity both invitro and in vivo,” Br. J. Cancer 88: 438-46 (2003).SR31747A alsoinhibited proliferation in vivo in the mouse xenograft model. Id. MurineEBP cDNA overexpression in CHO cells increased resistance of these cellsto SR31747A-induced inhibition of proliferation. Labit-Le Bouteiller, C.et al., “Antiprolifertive effects of SR31747A in animal cell lines aremediated by inhibition of cholesterol biosynthesis at the sterolisomerase step,” Eur. J. Biochem. 256: 342-49 (1998)),

Tamoxifen, inhibited SR31747 binding in a competitive manner and inducedthe accumulation of Δ8-sterols, while Emopamil, a high affinity ligandof human sterol isomerase, and verapamil, another calciumchannel-blocking agent, are inefficient in inhibiting SR31747 binding toits mammalian target, suggesting that their binding sites do notoverlap. Paul, R. et al., “Both the immunosuppressant SR31747 and theantiestrogen tamoxifen bind to an emopamil-insensitive site of MammalianΔ8-Δ7 sterol isomerase,” J. Pharmacol. Exptl Thera. 285(3): 1296-1302(1998)). Some drugs, e.g., cis-flupentixol, trifluoroperazine,7-=ketocholestanol and tamoxifen, inhibit SR31747 binding only withmammalian EBP enzymes, whereas other drugs, e.g., haloperidol andfenpropimorph, are more effective with the yeast enzyme than with themammalian ones. Id.

Example 3. Experiments Showing that Cholesterol Synthesis Enzymes areDownregulated by 4C12

FIG. 12A is a bar graph showing cholesterol (pg/cell), and triglyceride(pg/cell) levels in Mut6 cells treated with compound 4C12 for 24 hoursand 48 hours, versus a negative control (vehicle only). The figures showthat in the presence of compound 4C12, cholesterol level decreases, andtriglyceride level increases. FIG. 12B is a bar graph of relative mRNAvs. enzymes of cholesterol synthesis showing that genes for cholesterolsynthesis enzymes are down-regulated by 4C12. Mut6 cells were treatedwith compound 4C12 for 24 hours and then mRNA levels forHydroxymethylglutaryl-CoA synthase(Hmgcs);3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr); acetoacetyl-CoAsynthetase (AACS); Delta(24)-sterol reductase (Dhcr24);7-dehydrocholesterol reductase (Dhcr7), Sterol C5-desaturase (Sc5d);Squalene synthase (SS); and farnesyl pyrophosphate (FPP) synthase (FPPS)were determined.

In FIG. 13(a)-(e) Mut6 cells were treated with compound 4C12 for 2, 9,16, 24, and 48 hr, respectively, and their cholesterol gene profilecompared to a DMSO control. The data shows that compound 4C12 inhibitsSrebp2 target genes, and not Srebp1 target genes. (f) Western blot. Mut6cells were treated with vehicle only (−) or with compound 4C12 (+) for 7hr, 13 hr and 23 hr. Cells were collected, lysed in SDS buffer,subjected to SDS PAGE, and cell proteins transferred to a membrane by astandard protocol. The membrane was washed, treated with antibodies toSREBP1, SREBP2 and a positive control (Cadherin), rewashed and boundantibodies then revealed. The blots showed that compound 4C12 waseffective to decrease SREBP2 protein.

Example 4: Experiments to Determine Whether Decrease of Cholesterol isFunctionally Important in Compound 4C12-Induced Cell Death

FIGS. 14A and 15(a) are a bar graph plotting ATP activity (a measure ofviability) for Mut6 cells treated with compactin/mevastatin, compound4C12, and the combination of compactin/mevastatin+compound 4C12, versusa negative control (vehicle only). The results show that the combinationof compactin/mevastatin and compound 4C12 exert an effect greater thaneach does alone. FIG. 13B illustrates statins' inhibitory effect on themevalonate arm of the cholesterol biosynthesis pathway.

FIG. 15(b) is a bar graph showing ATP activity (a measure of viability)vs. concentration of cholesterol (μM)—Mut6 cells were treated withcompound 4C12 versus a cells treated with DMSO (negative control).Addition of cholesterol inhibits compound 4C12-induced cell-death. 15(c)shows relative ATP activity (y-axis, a measure of viability) vs.compound 4C12 concentration (nM (x axis). Addition of SREBP2 byknock-down of Insig1/Insig2 makes Mut6 cells less sensitive to compound4C12.

FIG. 16 is a bar graph of relative mRNA level for cholesterolbiosynthesis pathway target genes Hmgcs, Hmgcr, FPPS, LDLR; and SREBP1cin mouse embryo fibroblasts (left) and astrocytes (right) treated withcompound 4C12 or dmso (negative control). The figure shows that Srebp2target genes were not decreased by compound 4C12 in MEFs and Astrocytes.

FIG. 17 compares cholesterol level (pg/cell) (Left) and triglyceridelevel (pg/cell) (Right) in Mut6 cells and in MEF cells treated withcompound 4C12 for 24 hours and 48 hours to a negative control (vehicle).The results show that the observed decrease in cholesterol by compound4C12 is specific to Mut6 tumor cells, and that Mut6 cells have a muchlower basal level of cholesterol and triglycerides.

FIG. 18A is a bar graph showing the effect of inhibition of cholesterolbiosynthesis pathway genes Hmgcs; Hmgcr, AACS, Dhcr24, Dhcr7, Sc5d, SS,FPPS, LDLR; and SREBP2 in Mut6 cells treated with DMSO or with compound4C12 for 16 hours. FIG. 18B is a bar graph showing effect on level ofABCa1 of treating Mut6 cells with compound 4C12 for 16 hours versus aDMSO negative control.

FIG. 19 shows relative ATP level (y-axis, a measure of viability) vs.concentration of compound 4C12 (nM) for various primary patient derivedglioblastoma cell lines (top left and top right), and for HeLa(cervical), HT-29 (colon), 435 (breast), 549 (lung), MCF7 (breast),HCC38 (breast), Daoy (medulloblastoma (brain) cancer cell lines, andmouse embryonic fibroblast (MEF) cells in the presence (bottom left) andabsence (bottom right) of serum.

While the present invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A compound according to Formula I-g

wherein R₁ is selected from the group consisting of

n=0, 1, or 2; m=0 or 1; R₂ and R₃ can be attached at any availableposition on the aromatic ring and are independently selected from thegroup consisting of H, D, F, Cl, CF₃, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆ hydroxyalkyl, cycloalkyl, OR, N(R)₂, NO₂, N₃, NH—C(O)—R,CN, C(O)R, C(O)OR, C(O)N(R)₂, SR, alkylacyl, and arylacyl; Each R isindependently selected from the group consisting of H, C₁₋₃ alkyl,propargyl, and phenyl; R₄ and R₅ are independently selected from thegroup consisting of H, C₁₋₆ alkyl optionally substituted with N(R)₂,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ hydroxyalkyl, phenyl optionallysubstituted with R₃, and CH₂OC(O)-phenyl optionally substituted with R₃;R₆ and R₇ are independently selected from the group consisting of H,C₁₋₃ alkyl, and C(O)OMe₃; such that all possible stereoisomers,including optically active isomers, are included whenever stereogeniccenters are present; or a pharmaceutically acceptable salt, prodrug,active metabolite, or solvate thereof.
 2. A compound of claim 1according to Formula I-g

wherein R₂ and R₃ can be attached at any available position on thearomatic ring and are independently selected from the group consistingof H, D, F, Cl, CF₃, C₁-C₃ alkyl, C₂-C₃ alkenyl, C₂-C₃ alkynyl, C₁-C₃hydroxyalkyl, C₄-C₆ cycloalkyl, OR, N(R)₂, NO₂, N₃, NH—C(O)—R, CN,C(O)R, C(O)OR, C(O)N(R)₂, and SR; Each R is independently selected fromthe group consisting of H, C₁₋₃ alkyl, propargyl, and phenyl; such thatall possible stereoisomers, including optically active isomers, areincluded whenever stereogenic centers are present; or a pharmaceuticallyacceptable salt, prodrug, active metabolite, or solvate thereof.
 3. Apharmaceutical composition comprising a therapeutic amount of thecompound according to claim 1, and a pharmaceutically acceptablecarrier.
 4. A compound selected from the group consisting of:

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present; or apharmaceutically acceptable salt, prodrug, active metabolite, or solvatethereof.
 5. A compound selected from the group consisting of

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present; or apharmaceutically acceptable salt, prodrug, active metabolite, or solvatethereof.
 6. A compound selected from the group consisting of:

such that all possible stereoisomers, including optically activeisomers, are included whenever stereogenic centers are present; or apharmaceutically acceptable salt, prodrug, active metabolite, or solvatethereof.
 7. A pharmaceutical composition comprising a therapeutic amountof the compound according to any one of claims 4-6 and apharmaceutically acceptable carrier.